Structural insights into tail-anchored protein binding and membrane insertion by Get3

Bozkurt, Günes; Stjepanović, Goran; Vilardi, Fabio; Amlacher, Stefan; Wild, Klemens; Bange, Gert; Favaloro, Vincenzo; Rippe, Karsten; Hurt, Ed; Dobberstein, Bernhard; Sinning, Irmgard

doi:10.1073/pnas.0910223106

Cited by 99 publications

(165 citation statements)

References 51 publications

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“…We conclude that the role of Bat3 is most likely restricted to TA proteins that are TRC40 clients (Colombo et al, 2009;Rabu et al, 2009;Rabu et al, 2008;Stefanovic and Hegde, 2007). Although the peak of integration-competent Sec61b chains co-migrate with the bulk of TRC40, strongly supporting a role in promoting membrane integration (Bozkurt et al, 2009;Favaloro et al, 2008;Stefanovic and Hegde, 2007), the bulk of Bat3 comigrates with a pool of Sec61b chains that are poorly membrane integrated. Thus, although Bat3 can influence the fate of TAprotein substrates of the TRC40 pathway, its function might be spatially and temporally distinct from the TRC40-mediated delivery step that precedes membrane integration (Bozkurt et al, 2009;Favaloro et al, 2010).…”

Section: Discussionmentioning

confidence: 95%

“…TRC40/Get3 forms a stable association with newly synthesised TA proteins (Favaloro et al, 2008;Stefanovic and Hegde, 2007), and functions at a late stage of the ER-delivery process (Bozkurt et al, 2009). We investigated the relationship of Bat3 with newly synthesised Sec61b-OPG chains generated by cell-free translation.…”

Section: Bat3 and Trc40 Associate With Distinct Populations Of Sec61bmentioning

confidence: 99%

“…Furthermore, several recent studies of Get3 provide structural insights into the mechanisms that underlie its substrate binding and release, and provide models for how ATP binding and hydrolysis might influence these steps (Bozkurt et al, 2009;Hu et al, 2009;Mateja et al, 2009;Suloway et al, 2009). Studies of S. cerevisiae have shown that several components function in concert with Get3: Get1 and Get2 are ERlocalised membrane receptors for the GET pathway of TA-protein delivery (Schuldiner et al, 2008), whereas Get4 and Get5 are cytosolic components that appear to act in concert with Get3 before membrane delivery (Jonikas et al, 2009;Rabu et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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Bat3 promotes the membrane integration of tail-anchored proteins

Leznicki

Clancy

Schwappach

et al. 2010

Journal of Cell Science

120

184

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SummaryThe membrane integration of tail-anchored proteins at the endoplasmic reticulum (ER) is post-translational, with different tail-anchored proteins exploiting distinct cytosolic factors. For example, mammalian TRC40 has a well-defined role during delivery of tail-anchored proteins to the ER. Although its Saccharomyces cerevisiae equivalent, Get3, is known to function in concert with at least four other components, Get1, Get2, Get4 and Get5 (Mdy2), the role of additional mammalian proteins during tail-anchored protein biogenesis is unclear. To this end, we analysed the cytosolic binding partners of Sec61b, a well-defined substrate of TRC40, and identified Bat3 as a previously unknown interacting partner. Depletion of Bat3 inhibits the membrane integration of Sec61b, but not of a second, TRC40-independent, tail-anchored protein, cytochrome b5. Thus, Bat3 influences the in vitro membrane integration of tail-anchored proteins using the TRC40 pathway. When expressed in Saccharomyces cerevisiae lacking a functional GET pathway for tail-anchored protein biogenesis, Bat3 associates with the resulting cytosolic pool of non-targeted chains and diverts it to the nucleus. This Bat3-mediated mislocalisation is not dependent upon Sgt2, a recently identified component of the yeast GET pathway, and we propose that Bat3 either modulates the TRC40 pathway in higher eukaryotes or provides an alternative fate for newly synthesised tail-anchored proteins.

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Section: Discussionmentioning

confidence: 95%

Section: Bat3 and Trc40 Associate With Distinct Populations Of Sec61bmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Bat3 promotes the membrane integration of tail-anchored proteins

Leznicki

Clancy

Schwappach

et al. 2010

Journal of Cell Science

120

184

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“…The structures were manually built with Coot (19) and refined with Refmac 5 (20) and Phenix (21). Refinement statistics are given in supplemental (22,23). Amide HX was initiated by a 20-fold dilution of 100 pmol of either FtsY/NG/NGϩ1 (apo) or FtsY/ NG/NGϩ1 with an excess of nucleotides into D 2 O buffer containing 20 mM Hepes (pD 7.5), 200 mM NaCl, 10 mM MgCl 2 , and 10 mM KCl at 30°C.…”

Section: Methodsmentioning

confidence: 99%

Lipids Trigger a Conformational Switch That Regulates Signal Recognition Particle (SRP)-mediated Protein Targeting

Stjepanović

Kapp

Bange

et al. 2011

Journal of Biological Chemistry

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Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.The biogenesis of most membrane proteins and many secretory proteins depends on the signal recognition particle (SRP) 4 and the SRP receptor (SR). SRP binds to N-terminal signal sequences of nascent polypeptide chains at the ribosome (ribosome-nascent chain complexes (RNCs)) and acts as an adaptor between the ribosome and the membrane-embedded translocation channel (1-3). Interaction of SRP with SR (FtsY in bacteria and archaea and SR␣ in eukarya) specifies the target membrane and allows for the precise coordination of RNC release from SRP and its transfer to the translocation channel. Protein targeting critically depends on the two homologous GTPases present in SRP and SR forming a heterodimer (4). GTP binding to SRP and SR is required for heterodimer formation and GTP hydrolysis triggers the dissociation of the SRP-SR complex which resets the SRP system for a new round of translocation (5). Although FtsY does not contain a hydrophobic, transmembrane sequence, it was shown to be almost exclusively localized at the membrane (6). FtsY contains three domains: an N-terminal negatively charged A domain of unknown structure and function and the highly conserved N and G domains that form a structural and functional unit, the NG domain (7, 8) (see Fig. 1A). The A domain acts as negative regulator of the FtsY GTPase in a lipid-free environment (9) and was suggested to participate in membrane interaction of FtsY by its N-terminal region (10). However, the A domain is not essential in Escherichia coli as a truncation variant (termed NGϩ1) is functional in vivo (11). It was shown that the FtsY GTPase is activated by anionic phospholipids (9) and that the membrane interaction of FtsY is crucial for the release of RNCs from the SRP-FtsY complex (12, 13), which was confirmed recently (14). Membrane interaction of E. coli FtsY depends on a conserved motif at the N terminus of the N domain, referred to as membrane-targeting sequence (MTS) (15). Recently, strong genetic ...

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“…Even with AMPPNP bound, targeting and insertion from wild-type Get3•TA was efficient (compare black lines in Fig. 5 A vs. G); thus, ATP hydrolysis per se is not required for membrane targeting (8). Wild-type AMPPNP• Get3•TA and ATP.…”

Section: -H)mentioning

confidence: 99%

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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Structural insights into tail-anchored protein binding and membrane insertion by Get3

Cited by 99 publications

References 51 publications

Bat3 promotes the membrane integration of tail-anchored proteins

Bat3 promotes the membrane integration of tail-anchored proteins

Lipids Trigger a Conformational Switch That Regulates Signal Recognition Particle (SRP)-mediated Protein Targeting

A protean clamp guides membrane targeting of tail-anchored proteins

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