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

Abstract

Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including membrane fusion, protein translocation, and apoptosis. The ATPase Get3 (Asna1, TRC40) was identified recently as the endoplasmic reticulum targeting factor of TA proteins. Get3 consists of an ATPase and alpha-helical subdomain enriched in methionine and glycine residues. We present structural and biochemical analyses of Get3 alone as well as in complex with a TA protein, ribosome-associated membrane protein 4 (Ramp4). The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion. Amide proton exchange mass spectrometry shows that the alpha-helical subdomain of Get3 displays considerable flexibility in solution and maps the TA protein-binding site to the alpha-helical subdomain. The non-hydrolyzable ATP analogue AMPPNP-Mg(2+)- and ADP-Mg(2+)-bound crystal structures representing the pre- and posthydrolysis states are both in a closed form. In the absence of a TA protein cargo, ATP hydrolysis does not seem to be possible. Comparison with the ADP.AlF(4)(-)-bound structure representing the transition state (Mateja A, et al. (2009) Nature 461:361-366) indicates how the presence of a TA protein is communicated to the ATP-binding site. In vitro membrane insertion studies show that recombinant Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner. Although ATP hydrolysis is not required for Ramp4 insertion per se, it seems to be required for efficient insertion. We postulate that ATP hydrolysis is needed to release Get3 from its receptor. Taken together, our results provide mechanistic insights into posttranslational targeting of TA membrane proteins by Get3.

Fig. 1.

Overall structure of Get3. (A) AMPPNP-Mg²⁺-bound structure of C. therm. Get3. The dimer is in the closed state. N- and C-termini, the P-loop, swI, swII, and the Zn ion and secondary structure elements mentioned in the text are labeled. Coloring of secondary structure elements is done in a ramp from blue (N-terminus) to red (C-terminus). (B) ADP-Mg²⁺-bound structure of C. therm. Get3. The α-helical (green) and ATPase (blue) subdomains for the 2 chains are colored in similar shades. (C) Overall changes in the α-helical subdomains in different nucleotide states. Monomeric Get3 is shown with AMPPNP-Mg²⁺ of C. therm. (Left; same for ADP-Mg²⁺) and with ADP·AlF₄⁻ of S. cerevisiae (Right; PDB code 2woj). The α-helical and ATPase subdomains are colored in green and gray, respectively. In the AMPPNP-Mg²⁺-bound (and ADP-Mg²⁺-bound) states, the α-helical subdomains are only partially folded.

Fig. 2.

Localization of Ramp4-induced changes in Get3 using HX-MS. Ramp4-dependent changes in HX kinetics of Get3 segments are projected onto the crystal structure. Get3 secondary structure is colored according to the deuteron incorporation shown in Fig. S3 for Get3 alone (Left) and the Get3–Ramp4 complex (Right): blue, 0–20%; cyan, 20–40%; yellow, 40–60%; orange, 60–80%; and red, 80–100%. It should be noted that helix α7 gets significantly stabilized.

Fig. 3.

Functional characterization of the Get3–Ramp4 complex. (A) Membrane insertion and glycosylation of Ramp4. The chimeric C. therm. Get3-mammalian–Ramp4op complex (for definition of Ramp4op, see SI Materials and Methods) was incubated in the presence of ATP without (lane 1) or with (lanes 2 to 4) RMs. The mixtures were separated into a supernatant (Sn) fraction and a pellet (P) fraction. Endoglycosidase H (EndoH) was added to one-half of the pellet fraction (lane 4). (B) Nucleotide requirements for membrane insertion of Ramp4op. (Upper) Get3–Ramp4op complexes were incubated with RMs in the absence or presence of adenosine nucleotides as indicated. (Lower) Amounts of glycosylated Ramp4op observed with different nucleotides are shown. (C) Dependence of Ramp4op membrane insertion on a membrane proteinaceous factor. Get3–Ramp4op complex was incubated with RMs (lane 2) or with trypsin-treated RMs (T-RMs; lane 3) in presence of ATP.

Fig. 4.

Nucleotide-binding site of AMPPNP-Mg²⁺-bound Get3. (A) Key interactions in the nucleotide binding site in cis. Important elements such as the A-loop, P-loop, swI, and swII are labeled. AMPPNP and side chains of interacting amino acids are shown in sticks, Mg²⁺ as a wheat-colored sphere, and water molecules as gray spheres. (B) Key interactions in the nucleotide binding site in trans. Lys-35 from the P-loop and Glu-243 from helix α10 interact with AMPPNP in trans. Secondary structure elements belonging to the second monomer are indicated by a single quotation mark, such as α10′ and P-loop', in Figs. 4 and 5.

Fig. 5.

Communication between the TA protein and the nucleotide-binding sites in Get3. The Get3 prehydrolysis state (Left, AMPPNP-Mg²⁺) is compared with the transition state (Right, ADP·AlF₄⁻, PDB ID code 2woj). (A) Active site closure viewed from the TA binding site. In the AMPPNP-Mg²⁺-bound structure, helices α6 and α7 pack loosely against each other, leaving the nucleotide solvent exposed. In the transition state, they close tightly over the nucleotide binding site. (B) Differences in the α-helical subdomain and in nucleotide-binding motifs between the prehydrolysis and the transition states are shown with the following changes observed in the transition state: helix α6 rotates around 25°, and helix α7, swI, swII, and helix α10 move toward the nucleotide. Helices α6 and α7 interact by means of a hydrophobic interface (HI). (C) Detailed view of the active site. The closing up in the transition state is clearly visible. The catalytic water (W_Cat) can be placed by the conserved aspartate from swI only in the transition state.

Fig. 6.

Model for TA binding and insertion. In the cytosol, the nucleotide-free (and ADP-bound) Get3 dimer is open. ATP binding to Get3 drives closure of the dimer (step 1) and allows TA protein binding. TA binding induces the fully closed hydrolysis-competent state (step 2). Get1/2 receptor docking occurs before or after ATP hydrolysis, with ADP and inorganic phosphate staying trapped in the closed active site (step 3). After TA protein release, inorganic phosphate is released and Get3 dissociates from the Get1/2 receptor (step 4). The Get3 dimer adopts an open structure on release of Mg²⁺ (step 5).