Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue

Abstract

Efficient delivery of membrane proteins is a critical cellular process. The recently elucidated GET (Guided Entry of TA proteins) pathway is responsible for the targeted delivery of tail-anchored (TA) membrane proteins to the endoplasmic reticulum. The central player is the ATPase Get3, which in its free form exists as a dimer. Biochemical evidence suggests a role for a tetramer of Get3. Here, we present the first crystal structure of an archaeal Get3 homologue that exists as a tetramer and is capable of TA protein binding. The tetramer generates a hydrophobic chamber that we propose binds the TA protein. We use small-angle X-ray scattering to provide the first structural information of a fungal Get3/TA protein complex showing that the overall molecular envelope is consistent with the archaeal tetramer structure. Moreover, we show that this fungal tetramer complex is capable of TA insertion. This allows us to suggest a model where a tetramer of Get3 sequesters a TA protein during targeting to the membrane.

Figure 1

The structure of MjGet3. (A) The overall structure of MjGet3 in cartoon representation with each subunit coloured differently and one subunit colour ramped blue to red from N- to C-terminus with approximate measurements on the side. Nucleotides are represented as sticks and ions as spheres. (B) 90° rotation relative to (A). (C) A monomer of MjGet3 colour ramped as in (A) overlaid with ScGet3 (2WOJ-A) shown in grey. Helix 1 is not resolved in MjGet3 and helix 13 is not obviously present. Dashed line connects helix 8 to helix 9. (D) A view of the nucleotide binding pocket highlighting hydrolase features: P-loop (cyan), switch I (purple), switch II (blue) and A-loop (orange). The opposing subunit in tan. ADP is in sticks coloured by atoms. Mg²⁺ as green sphere. 2F₀−F_C density for the nucleotide is shown as a blue mesh contoured at 2.5σ. (E) A split-view comparison of the dimers of MjGet3 (lavender) and ScGet3 (2WOJ—green). The dimers each have two-fold symmetry in the views shown. For clarity, only half of each structure from the overlay is shown to give a direct comparison. MjGet3 on the left and ScGet3 on the right. Coloured helices are 4/5 (teal), 6 (purple) and 7/8/9 (light orange). (F) A 90° rotation relative to (E).

Figure 2

Central cavity. (A) Surface representation of MjGet3 cut through the middle. Residues coloured based on type: positive charge (blue), negative charge (red) and hydrophobic (green). (B) Similarly to Figure 1A rotated 90° forward showing the cage walls with the foreground removed. (C) External view of the cavity in surface representation coloured based on electrostatic potential (negative—red to positive—blue). Holes indicated by arrow. (D) External view of the central cavity highlighting mutants from previous studies, inset shows zoom in region coloured similarly to Figure 1E. Mutants resulting in a negative growth phenotype by Suloway et al (2009) are shown in green. Mutants from Mateja et al (2009) are coloured according to loss of nucleotide hydrolysis (red), TA binding (blue) or both (purple).

Figure 3

TA protein binding by Get3. (A) Diagram of recombinantly expressed complex showing the two affinity tags used for purification (MBP, maltose binding protein). Position of thrombin cleavage site indicated. (B) Coomassie-stained SDS–PAGE of Get3/TA protein complexes from various species purified by recombinant co-expression. (C) Sequence of Sbh1 fusion to MBP with residues from truncations indicated. The TMD is in bold. (D) SDS–PAGE and western blot of the ScGet3/Sbh1 truncation complexes pre- and post-thrombin cleavage with αMBP antibody against the MBP–Sbh1 fusion. Accessibility of protease site results in a shift of the MBP–Sbh1 fusion to a lower MBP band. Residues numbers of Sbh1 are indicated. Figure source data can be found in Supplementary data.

Figure 4

Oligomeric state of Get3. (A) SEC of Get3 from S. cerevisiae (blue) and three archaeal species: M. jannaschii (green), M. maripaludis (red) and T. kodakaraensis (cyan). Tetramers and dimers eluted around 12 and 14 ml, respectively. (B) SEC run on different columns of ScGet3 after affinity purification (solid line). The tetramer peak was pooled and rerun (dashed line). (C) The three-helix bundle that stabilizes Get3 tetramers coloured as in Figure 2B with mutated residues highlighted. (D) SEC of MjGet3 in the absence (green) and presence (black) of 1% β-OG and a sample where detergent was dialysed out (dashed). Detergent shifts the peak to a volume corresponding to a dimer (13 ml). After dialysis, the peak shifts back to a tetramer (11 ml). (E) SEC of MjGet3 mutants. The 192–202 GAAG trace (yellow) corresponds to a deletion of helix 8.

Figure 5

Functional studies of Get3/TA protein tetramer complex and SEC-MALLS. (A) In-vitro membrane integration of an MBP fusion to Sbh1 with a C-terminal opsin tag into microsomes purified from S. cerevisiae Δget3 by a purified ScGet3/MBP–Sbh1-op complex. A western blot against MBP of in-vitro translocation assays into trypsinized yeast microsomes (T-YM—lane 1), in the presence of EDTA (lane 2), standard in-vitro translocation conditions before (lane 3) and after EndoH treatment (lane 4) and MBP–Sbh1 purified without Get3 (lane 5). Star indicates MBP–Sbh1-op and the arrow points to band shifted by glycosylation of MBP–Sbh1-op after membrane integration. (B) Molecular weights of Get3 and Get3/TA protein complexes measured by SEC coupled to MALLS. Traces of differential index of refraction (dn/dc) and calculated molecular weights are shown. Figure source data can be found in Supplementary data.

Figure 6

Size and shape of Get3/TA protein complexes. (A) Pair-distribution functions from bioSAXS of MjGet3 (blue), ScGet3/Ysy6 (green) and ScGet3/Sbh1_47–82 (salmon). (B) Calculated envelope of MjGet3 from bioSAXS data (blue mesh) with MjGet3 coordinates fit to the overall envelope. Insets are three views 90° rotated around the vertical and horizontal axis. Lines are drawn through the widest point of each lobe and a crossing angle is calculated along the long axis. Top left inset shows the crossing angle for the structure alone. (C) Similarly to (B) for the envelope of ScGet3/Ysy6 complex (green mesh). (D) Same as (C) for the envelope of the ScGet3/Sbh1_47–82 complex (salmon mesh).

Figure 7

Model for Get3 TA targeting.