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Structure of the Get3 targeting factor in complex with its membrane protein cargo

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Science  06 Mar 2015:
Vol. 347, Issue 6226, pp. 1152-1155
DOI: 10.1126/science.1261671

How to GET to the right membrane

Membrane proteins with a hydrophobic transmembrane domain (TMD) play critical roles in virtually all aspects of cell physiology. After it has been synthesized in the cytosol, this TMD must be targeted to and inserted into the correct membrane. The GET pathway is one of two targeting pathways to the endoplasmic reticulum conserved across all eukaryotes. It is not clear how the central targeting factor, Get3, recognizes a TMD to shield it from aggregation until it is successfully inserted into the membrane. Now, Mateja et al. show that the functional targeting complex comprises a Get3 dimer bound to a single TMD. The helical hydrophobic TMD binds deep within a large hydrophobic groove in the Get3 dimer. This groove closes slightly upon TMD binding, forming a dynamic “lid” over the mouth of the groove.

Science, this issue p. 1152

Abstract

Tail-anchored (TA) proteins are a physiologically important class of membrane proteins targeted to the endoplasmic reticulum by the conserved guided-entry of TA proteins (GET) pathway. During transit, their hydrophobic transmembrane domains (TMDs) are chaperoned by the cytosolic targeting factor Get3, but the molecular nature of the functional Get3-TA protein targeting complex remains unknown. We reconstituted the physiologic assembly pathway for a functional targeting complex and showed that it comprises a TA protein bound to a Get3 homodimer. Crystal structures of Get3 bound to different TA proteins showed an α-helical TMD occupying a hydrophobic groove that spans the Get3 homodimer. Our data elucidate the mechanism of TA protein recognition and shielding by Get3 and suggest general principles of hydrophobic domain chaperoning by cellular targeting factors.

Integral membrane proteins contain hydrophobic transmembrane domains (TMDs) that must be shielded from the cytosol until their insertion into the lipid bilayer. Whereas most eukaryotic membrane proteins are cotranslationally targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP) (1), tail-anchored (TA) membrane proteins are posttranslationally targeted by the cytosolic factor Get3 (27). This conserved adenosine triphosphatase (ATPase) changes conformation in a nucleotide-regulated manner (812) to bind TMDs in the cytosol and release them at its ER membrane receptor (6, 1316).

Assembly of the Get3-TA targeting complex requires “pretargeting” factors that mediate loading onto Get3 (17, 18). This pathway begins with TA protein in complex with the chaperone Sgt2. The Get4-Get5 scaffolding complex then recruits Sgt2 via Get5, while Get4 recruits ATP-bound Get3 (19). A hand-off reaction within this complex results in transfer of TA protein from Sgt2 to Get3. TA substrate-loaded Get3 then dissociates from Get4 (2022), resulting in a targeting complex whose architecture and stoichiometry have been debated (812, 2023).

To define the physiologically relevant Get3 targeting complex, we recapitulated its assembly in vitro, using purified recombinant factors at in vivo concentrations (Fig. 1A). Translation of radiolabeled TA protein in the presence of SGTA (the mammalian homolog of Sgt2) produced a stable complex detectable by chemical cross-linking (fig. S1). The TA protein remained associated with SGTA upon addition of either Get4-Get5 or Get3, but released efficiently when both factors were added (Fig. 1B). Correspondingly, Get3 efficiently acquired substrate from SGTA only when Get4-Get5 was present.

Fig. 1 Reconstitution of physiologic TA protein targeting complex assembly.

(A) Experimental strategy. (B) SGTA-TA or Get3-TA complexes (figs. S1 and S3) at 1 μM were incubated with 1 μM of the indicated proteins, followed by amine-reactive cross-linking. Reactions were analyzed by SDS–polyacrylamide gel electrophoresis and Coomassie blue staining to detect the input proteins (top) or autoradiography to detect the 35S-labeled TA protein cross-links (bottom). (C) Reactions as in (B) were monitored by sulfhydryl-reactive cross-linking for TA protein release from SGTA (bottom). Reactions contained 0.5 μM of each factor, except lanes 4 and 5, which contained Get4-Get5 at 0.1 and 0.2 μM, respectively. Asterisks next to Get3 or SGTA indicate point mutants that disrupt interactions with Get4 or Get5, respectively. (D) Products of the indicated transfer reactions were incubated with yeast rough microsomes (yRM) and analyzed for insertion. (E) Sucrose gradient size analysis of Get3-TA complex formed by Get4-Get5–dependent loading from SGTA (red). Free, dimeric Get3 (gray) and E. coli–produced tetrameric Get3-TA substrate complex (black) are shown for comparison (fig. S5). Peak fractions containing substrate (red) were analyzed directly or after cross-linking and immunoprecipitation for Get3 to specifically detect Get3-TA complexes (bottom panel).

The transfer reaction was rapid and unidirectional: Once substrate released from SGTA, it did not rebind (fig. S2). Likewise, substrate preloaded directly on Get3 (fig. S3) did not effectively transfer to SGTA (Fig. 1B). Structure-guided mutations disrupting either the SGTA-Get5 interaction [SGTA(C38S)] (24) or the Get4-Get3 interaction [Get3(E253R)] (20) abolished substrate release from SGTA (Fig. 1C). Targeting complex produced via Get4-Get5 supported TA protein insertion into yeast ER microsomes (Fig. 1D), while an identical reaction containing SGTA(C38S) showed reduced insertion (Fig. 1D). Thus, the recombinant assembly system requires all factors and interactions of the early GET pathway and produces insertion-competent Get3-TA protein targeting complex.

Three lines of evidence suggested that functional targeting complex assembled via pretargeting factors consists of dimeric Get3 bound to TA protein. First, the targeting complex, containing a small (~10 kD) TA protein, had the same native size as purified Get3 dimer, and was clearly distinguishable from higher-order Get3 complexes (Fig. 1E). Such higher-order complexes, often seen when Get3 is coexpressed with TA protein in Escherichia coli (fig. S5) (8, 22, 23), were not observed even when the loading reaction contained 10-fold excess Get3 (fig. S4A). Second, titration of Get3 into the loading reaction showed no evidence of cooperativity (fig. S4B), arguing against its higher-order assembly during targeting complex formation. Third, size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) indicated that prior to loading, a single Get3 dimer is bound by two copies of the Get4-Get5 complex (fig S4C). Thus, TA protein is loaded onto dimeric Get3 to form a functional targeting complex.

To gain insight into how the TA protein is shielded by Get3 in this targeting complex, we sought to determine its structure. During physiologic targeting complex assembly, Get4 preferentially recruits and stabilizes adenosine 5′-triphosphate (ATP)–bound Get3 (19, 20, 22). To mimic this during recombinant expression in E. coli, we biased Get3 to the ATP-bound state via the D57N hydrolysis mutant (10). Coexpression of this mutant with TA protein resulted in a targeting complex that was homogeneously dimeric for Get3 by SEC-MALLS (fig. S6A) and comigrated with in vitro–assembled targeting complex on sucrose gradients (fig. S6B).

To facilitate crystallization, we generated a high-affinity synthetic antibody fragment (sAB) (25) that recognizes the closed (ATP-bound) conformation of Get3. Kinetic analysis revealed that this sAB binds with subnanomolar affinity to nucleotide-bound Get3, both in the presence and absence of TA protein (fig. S7). Thus, rather than inducing a large conformational change in Get3, the TA protein binds to a preorganized conformation that closely resembles the closed (ATP-bound) state.

Using this sAB, we crystallized Get3(D57N) in complex with the TMD of the yeast TA protein Pep12 (table S1). The structure reveals nucleotide-bound Get3 in a closed conformation with two sABs bound to equivalent sites on opposite faces of a Get3 homodimer (Fig. 2A); no higher-order Get3 oligomers are observed in the crystal (fig. S8). The closed conformation is nearly identical to that seen in previous Get3-ADP•AlF4 structures (~0.5 Å root mean square deviation), in which two helical subdomains form a composite hydrophobic groove proposed to bind the TMDs of TA proteins (8, 10).

Fig. 2 The helical TMD of a TA substrate binds deep within the composite hydrophobic groove of dimeric Get3.

(A) Overview of dimeric Saccharomyces cerevisiae Get3 bound to a truncated Pep12 TA substrate (magenta) and nucleotide (spheres), and sandwiched between two copies of an engineered sAB (gray). At right, a “side” view of the complex is shown with sABs removed for clarity. (B) Details of the interaction between the Pep12 TMD C terminus and a methionine-rich cluster at one end of the hydrophobic groove. Electron density is from a 2.05 Å 2FoFc map contoured at 1.0σ. (Amino acid abbreviations: F, Phe; I, Ile; L, Leu; and M, Met.) (C) Surface representations of the TA substrate-binding site, colored from least (white) to most (green) hydrophobic.

As is typical for the fungal Get3 crystal structures, electron density is weakest within these dynamic helical subdomains. Nevertheless, unaccounted helical density was visible within the hydrophobic groove in early unbiased maps (fig. S9). After refinement, we assigned this density to the Pep12 TMD (Fig. 2B and fig. S9), excluding the possibility that it corresponds to flexible regions of Get3 folding into the groove.

The Pep12 TMD binds to Get3 at the bottom of the composite hydrophobic groove (Fig. 2 and fig. S9), where it spans the dimer interface and stabilizes the closed conformation of Get3. The most ordered interactions are found at the ends of the TMD, where bulky hydrophobic side chains of the substrate contact groove residues including M97 (helix 4), L126 (helix 5), M143 and M146 (helix 6), L183, L186 and F190 (helix 7), and L216 and L219 (helix 9) (Fig. 2B). Consistent with their role in TMD binding, substitution of hydrophobic residues along helices 7 and 9 with polar or charged residues abolished Get3’s ability to induce TA protein release from SGTA (Fig. 3A).

Fig. 3 Dynamic shielding of the TMD.

(A) SGTA-TA complexes were prepared and subjected to transfer reactions with wild-type (WT) and mutant Get3 proteins as in Fig. 1C. LL-SS (L183S/L186S), LLL-SSS (L183S/L186S/L219S), and FL-DD (F190D/L216D) are hydrophobic groove mutants; E253R is a mutation that disrupts interaction with Get4. (Amino acid abbreviations: D, Asp; E, Glu; F, Phe; L, Leu; R, Arg; and S, Ser.) (B) “Top” and “side” views of Pep12 (magenta), Nyv1 (blue), and Sec22 (green) complexes superimposed on the free Get3 closed dimer structure (yellow; PDB code: 2woj). Relative to free Get3, the end of helix 7 extends and begins to curve inward over the substrate. (C) WT or benzophenone-containing (at the indicated positions) Get3-TA complexes were prepared as in fig. S3, and the dimer peak was subjected to ultraviolet (UV) cross-linking. Uncrosslinked TA protein and its adducts to one or two Get3 proteins are indicated. (D) “Side” views of the Get3 dimer, looking into the groove. In its transient empty state, Get3 is splayed apart, with two hydrophobic “half-sites” occupied by the helix 8 region. ATP binding drives Get3 into a closed conformation, which is captured by two copies of the Get4-Get5 complex. In this state, helix 8 is displaced, and the composite hydrophobic groove is now preorganized for substrate binding. After substrate transfer from Sgt2, the targeting complex is released. The helix 8 region now dynamically shields the substrate during transit to the ER membrane.

The Pep12 TMD buries ~1450 Å2 of hydrophobic surface area, distributed nearly evenly between the two Get3 subunits (Fig. 2C). This represents ~50% of the ordered hydrophobic surface area in the groove and is significantly greater than in the SRP54-signal peptide interaction, where ~360 Å2 of hydrophobic surface area become buried upon binding (26, 27). The availability of such a large surface area likely explains how Get3 can accommodate hydrophobic sequences of differing lengths and composition.

Using the same strategies, we also solved crystal structures of Get3(D57N) in complex with unrelated TMDs from Nyv1 and Sec22 (table S1). Density for these TMDs was less defined than for the Pep12 complex, but nevertheless sufficient to place helical TMDs (fig. S9). Like Pep12, these TMDs bind at the bottom of the hydrophobic groove, spanning the dimer interface (Fig. 3B). Thus, a single helix binding across the Get3 dimer represents the canonical mode of the Get3-TA substrate interaction.

Although much of the Get3 hydrophobic groove and substrate TMD are shielded in the targeting complexes, one surface of the TMD appears solvent exposed. Relative to previous closed Get3 structures, the groove in each substrate-bound complex is constricted at its apex where the ends of helix 7 curve inwards (Fig. 3B). Although the “TRC40-insert,” including helix 8, is poorly defined, we found by site-specific photo–cross-linking that this region (and residues in helix 6 and 7) directly contact the TA substrate (Fig. 3C and fig. S10). Thus, helix 8 likely functions as a dynamic “lid,” protecting the TMD from aggregation, while still allowing substrate release after recruitment to Get1 (Fig. 3D) (13, 14).

Our biochemical and structural analyses define the functional targeting complex as a Get3 homodimer bound to a single TA protein. Although higher-order Get3 assembly has been postulated to promote ATP hydrolysis (22), this appears unnecessary because dimeric targeting complex was functional for TA protein insertion, indicating that it had hydrolyzed its ATP (Fig. 1D). Consistent with this, the catalytic machinery is organized for hydrolysis in the targeting complex structures (fig. S11). The higher-order Get3 oligomers that form during oxidative stress (28) are structurally and functionally distinct.

The structure of the Get3-TA substrate targeting complex illustrates a common strategy for binding to hydrophobic cargo. Like Get3, the signal sequence–binding subunit of SRP (SRP54) captures substrates within a hydrophobic, methionine-rich groove presented on a helical scaffold (26, 27, 29). These scaffolds provide a large and intrinsically dynamic binding site that is not appreciably ordered by substrate capture. This likely confers the ability of Get3 and SRP54 to bind a variety of hydrophobic sequences—an essential property of both targeting systems. It will be of interest to determine whether these principles are shared by other TMD-binding factors, including SGTA and Bag6.

Supplementary Materials

www.sciencemag.org/content/347/6226/1152/suppl/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S12

Table S1

References (3037)

References and Notes

  1. Acknowledgments: We thank S. Shao for help with assay development; S. Koide for the phage library; S. Sidhu for the sAB expression vector; M. Kivlen for plasmids; F. Bezanilla, E. Perozo ,and J. Piccirilli for instrumentation; members of the Keenan, Hegde, and Kossiakoff labs for support; and the NE-CAT (24-ID-C) beamline staff at Advanced Photon Source for technical assistance. NE-CAT is supported by NIH grant P41 GM103403 and U.S. Department of Energy contract DE-AC02-06CH11357. Additional support was from the UK Medical Research Council (MC_UP_A022_1007 to R.S.H.), the NIH (U01 GM094588 and U54 GM087519 to A.A.K.; R01 GM086487 to R.J.K.), and the Searle Funds at The Chicago Community Trust for the Chicago Biomedical Consortium (to A.A.K. and R.J.K.). The Protein Data Bank (PDB) accession codes are 4XTR (Pep12), 4XVU (Nyv1), and 4XWO (Sec22).
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