Get3 ATPase Research Articles

Subunit cooperation in the Get1/2 receptor promotes tail-anchored membrane protein insertion.

The guided entry of tail-anchored protein (GET) pathway, in which the Get3 ATPase delivers an essential class of tail-anchored membrane proteins (TAs) to the Get1/2 receptor at the endoplasmic reticulum, provides a conserved mechanism for TA biogenesis in eukaryotic cells. The membrane-associated events of this pathway remain poorly understood. Here we show that complex assembly between the cytosolic domains (CDs) of Get1 and Get2 strongly enhances the affinity of the individual subunits for Get3•TA, thus enabling efficient capture of the targeting complex. In addition to the known role of Get1CD in remodeling Get3 conformation, two molecular recognition features (MoRFs) in Get2CD induce Get3 opening, and both subunits are required for optimal TA release from Get3. Mutation of the MoRFs attenuates TA insertion into the ER in vivo. Our results demonstrate extensive cooperation between the Get1/2 receptor subunits in the capture and remodeling of the targeting complex, and emphasize the role of MoRFs in receptor function during membrane protein biogenesis.

A Chaperone Lid Ensures Efficient and Privileged Client Transfer during Tail-Anchored Protein Targeting

Molecular chaperones play key roles in maintaining cellular proteostasis. In addition to preventing client aggregation, chaperones often relay substrates within a network while preventing off-pathway chaperones from accessing the substrate. Here we show that a conserved lid motif lining the substrate-binding groove of the Get3 ATPase enables these importantfunctions during the targeted delivery of tail-anchored membrane proteins (TAs) to the endoplasmic reticulum. The lid prevents promiscuous TA handoff to off-pathway chaperones, and more importantly, it cooperates with the Get4/5 scaffolding complex to enable rapid and privileged TA transfer from the upstream co-chaperone Sgt2 to Get3. These findings provide a molecular mechanism by which chaperones maintain the pathway specificity of client proteins in the crowded cytosolic environment.

Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting

Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C terminus, are post-translationally targeted to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 complex (Get4/5), which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3·Get4/5 complex. Here we report crystal structures of a Get3·Get4/5 complex from Saccharomyces cerevisiae at 2.8 and 6.0 Å that reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3·Get4/5 complex. Furthermore, we provide evidence that the Get3·Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limited electrostatically driven Get3·Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway.

Structural and functional characterization of ybr137wp implicates its involvement in the targeting of tail-anchored proteins to membranes.

Nearly 5% of membrane proteins are guided to nuclear, endoplasmic reticulum (ER), mitochondrial, Golgi, or peroxisome membranes by their C-terminal transmembrane domain and are classified as tail-anchored (TA) membrane proteins. In Saccharomyces cerevisiae, the guided entry of TA protein (GET) pathway has been shown to function in the delivery of TA proteins to the ER. The sorting complex for this pathway is comprised of Sgt2, Get4, and Get5 and facilitates the loading of nascent tail-anchored proteins onto the Get3 ATPase. Multiple pulldown assays also indicated that Ybr137wp associates with this complex in vivo. Here, we report a 2.8-Å-resolution crystal structure for Ybr137wp from Saccharomyces cerevisiae. The protein is a decamer in the crystal and also in solution, as observed by size exclusion chromatography and analytical ultracentrifugation. In addition, isothermal titration calorimetry indicated that the C-terminal acidic motif of Ybr137wp interacts with the tetratricopeptide repeat (TPR) domain of Sgt2. Moreover, an in vivo study demonstrated that Ybr137wp is induced in yeast exiting the log phase and ameliorates the defect of TA protein delivery and cell viability derived by the impaired GET system under starvation conditions. Therefore, this study suggests a possible role for Ybr137wp related to targeting of tail-anchored proteins.

Crystal structure of ATP-bound Get3-Get4-Get5 complex reveals regulation of Get3 by Get4.

Correct localization of membrane proteins is essential to all cells. Chaperone cascades coordinate the capture and handover of substrate proteins from the ribosome to their target membrane; yet the mechanistic and structural details of these processes remain unclear. Here we investigate the conserved GET pathway, in which the Get4-Get5 complex mediates the handover of tail-anchor (TA) substrates from the co-chaperone Sgt2 to the Get3 ATPase, the central targeting factor. We present a crystal structure of a yeast Get3-Get4-Get5 complex in an ATP-bound state, and show how Get4 primes Get3 into the optimal configuration for substrate capture. Structure-guided biochemical analyses demonstrate that Get4-mediated regulation of ATP hydrolysis by Get3 is essential to efficient TA protein targeting. Analogous regulation of other chaperones or targeting factors could provide a general mechanism for ensuring effective substrate capture during protein biogenesis.

The structure of a tail‐anchor membrane protein‐binding complex reveals the regulation of Get3 by Get4 (950.4)

Tail‐anchored (TA) membrane proteins are targeted to the endoplasmic reticulum via the post‐translational GET pathway. A critical step in this essential pathway is the transfer of TA substrates from the co‐chaperone Sgt2 to the Get3 ATPase, mediated by the hetero‐tetrameric complex Get4/Get5 (Get4/5). However, a major outstanding question is how Get4 binds to Get3 and regulates its activity. Here we report a 5.4 Å crystal structure of an ATP‐bound Get3‐Get4/5 complex from Saccharomyces cerevisiae (Sc), the largest pathway components solved to date. Our structure reveals the role of nucleotide in complex formation and the presence of two functionally distinct binding interfaces. Mutational analysis confirms that one interface is required for the specificity of binding, while the other interface is involved in the regulation of Get3 ATPase activity. Additional functional assays demonstrate that Get4/5‐mediated regulation of ATP hydrolysis by Get3 is essential for efficient TA protein targeting. Our work also supports a model for Get3/TA complex formation in which two opposing Get3 dimers are bridged by a single Get4/5 hetero‐tetramer. Finally, we present a 2.8 Å crystal structure of an apo ScGet3‐Get4/5 complex that provides evidence for a rapid initial binding state mediated by electrostatics. This work illustrates how Get4/5 regulates Get3, priming it for TA loading, a critical step in this important pathway.Grant Funding Source: NIH

SIMIBI twins in protein targeting and localization

Signal recognition particle, MinD and BioD (SIMIBI)-type nucleoside triphosphate-binding proteins are an ancient subfamily of nucleotide-binding proteins that serve in a wide range of cellular processes. Notably, this class comprises dimeric ATPases as well as GTPases (SIMIBI 'twins') and a subset of SIMIBI-type proteins, including SRP GTPases, MinD-type ATPases and the Get3 ATPase, is essential to protein targeting and localization. Here, we define common mechanistic principles and differences for these SIMIBI proteins.

Precise timing of ATPase activation drives targeting of tail-anchored proteins

The localization of tail-anchored (TA) proteins, whose transmembrane domain resides at the extreme C terminus, presents major challenges to cellular protein targeting machineries. In eukaryotic cells, the highly conserved ATPase, guided entry of tail-anchored protein 3 (Get3), coordinates the delivery of TA proteins to the endoplasmic reticulum. How Get3 uses its ATPase cycle to drive this fundamental process remains unclear. Here, we establish a quantitative framework for the Get3 ATPase cycle and show that ATP specifically induces multiple conformational changes in Get3 that culminate in its ATPase activation through tetramerization. Further, upstream and downstream components actively regulate the Get3 ATPase cycle to ensure the precise timing of ATP hydrolysis in the pathway: the Get4/5 TA loading complex locks Get3 in the ATP-bound state and primes it for TA protein capture, whereas the TA substrate induces tetramerization of Get3 and activates its ATPase reaction 100-fold. Our results establish a precise model for how Get3 harnesses the energy from ATP to drive the membrane localization of TA proteins and illustrate how dimerization-activated nucleotide hydrolases regulate diverse cellular processes.

Precise Timing of ATPase Activation Drives Targeting of Tail-Anchored Proteins

Hundreds of proteins are anchored on cellular membranes by a transmembrane domain (TM) at their extreme C-terminus. These ‘tail-anchored' (TA) proteins perform numerous essential cellular functions, yet their unique topology poses fundamental challenges to their proper localization. In eukaryotic cells, the highly conserved ATPase Get3 coordinates the efficient capture and delivery of TA proteins to the endoplasmic reticulum (ER). How Get3 uses its ATPase cycles to drive this fundamental process remains elusive. Here we define the Get3 ATPase cycle and show that ATP specifically induces conformational changes in Get3 that culminate in its ATPase activation through tetramerization. This activation is inhibited by the TA protein loading complex Get4/5 and is activated by the TA substrate, ensuring the precise timing of ATP hydrolysis during targeting. Our results provide an explicit model for how Get3 harnesses the energy from ATP to spatially and temporally coordinate the membrane localization of TA proteins.

Get1 Stabilizes an Open Dimer Conformation of Get3 ATPase by Binding Two Distinct Interfaces

Tail-anchored (TA) proteins are integral membrane proteins that possess a single transmembrane domain near their carboxy terminus. TA proteins play critical roles in many important cellular processes such as membrane trafficking, protein translocation, and apoptosis. The GET complex mediates posttranslational insertion of newly synthesized TA proteins to the endoplasmic reticulum membrane. The GET complex is composed of the homodimeric Get3 ATPase and its heterooligomeric receptor, Get1/2. During insertion, the Get3 dimer shuttles between open and closed conformational states, coupled with ATP hydrolysis and the binding/release of TA proteins. We report crystal structures of ADP-bound Get3 in complex with the cytoplasmic domain of Get1 (Get1CD) in open and semi-open conformations at 3.0- and 4.5-Å resolutions, respectively. Our structures and biochemical data suggest that Get1 uses two interfaces to stabilize the open dimer conformation of Get3. We propose that one interface is sufficient for binding of Get1 by Get3, while the second interface stabilizes the open dimer conformation of Get3.

Regulation of the Get3 ATPase Cycle

The cytosolic ATPase Get3/TRC40 mediates targeting of tail‐anchored (TA) membrane proteins to the endoplasmic reticulum (ER). Get3 functions as a molecular chaperone by binding to transmembrane domains of newly synthesized TA proteins and delivering them to a membrane docking complex in the ER. Previous work have shown that ATP binding and hydrolysis can regulate Get3's function and are essential for the targeting of TA proteins. However, the precise mechanisms by which this is accomplished remain unclear. Using a combination of mechanistic enzymology and biophysical methods, we delineate Get3's ATPase cycle and show that it is required at multiple stages during TA protein targeting. Our data complements previous structural studies and contributes to our understanding of how Get3 harnesses the energy of ATP hydrolysis to spatially and temporally coordinate the delivery of TA proteins to the ER membrane.

Ｇｅｔ複合体によるＴａｉｌ‐ａｎｃｈｏｒｅｄタンパク質の膜埋込みメカニズム

Tail anchored proteins, defined by a single transmembrane domain at C-terminus, are post-translationally inserted into the membrane by Get3 ATPase. Here we report the crystal structure of Get3 in ADP-bound and nucleotide free forms. Get3 forms an open dimer conformation, in which two subunits are linked by a Zn2+ ion. Together with the biochemical studies, we propose the ATP independent TA protein binding, and the membrane insertion by conformational change of Get3 coupled with ATP hydrolysis.

Model for eukaryotic tail-anchored protein binding based on the structure of Get3

The Get3 ATPase directs the delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER). TA-proteins are characterized by having a single transmembrane helix (TM) at their extreme C terminus and include many essential proteins, such as SNAREs, apoptosis factors, and protein translocation components. These proteins cannot follow the SRP-dependent co-translational pathway that typifies most integral membrane proteins; instead, post-translationally, these proteins are recognized and bound by Get3 then delivered to the ER in the ATP dependent Get pathway. To elucidate a molecular mechanism for TA protein binding by Get3 we have determined three crystal structures in apo and ADP forms from Saccharomyces cerevisae (ScGet3-apo) and Aspergillus fumigatus (AfGet3-apo and AfGet3-ADP). Using structural information, we generated mutants to confirm important interfaces and essential residues. These results point to a model of how Get3 couples ATP hydrolysis to the binding and release of TA-proteins.