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    • Initial structural and biochemical work showed that Get form

    2021-11-23

    Initial structural and biochemical work showed that Get3 forms an obligate dimer whose conformation is regulated by the bound nucleotide. Analogous to SRP and SR, Get3 contains a P-loop nucleotide hydrolase domain in which the bound ATPs face one another at the dimer interface (Figure 3A, top right structure) 61, 62, 63. Also analogous to the SRP and SR NG-domains, the ATPase domain of Get3 is structurally and functionally coupled to a helical domain (Figure 3A, top structures). Nucleotide binding adjusts the Get3 dimer interface, which is amplified into larger movements of its helical domains. This leads to various conformations, from more ‘open’ states in apo-Get3 in which the helical domains are separated to more ‘closed’ states in Get3 bound to nonhydrolyzable ATP analogs, such as AMPPNP and ADP–AlF4–, in which the helical domains are close together (Figure 3A, Step 1) 61, 62, 63. Importantly, multiple hydrophobic residues in the helical domains are brought into a contiguous groove in the ‘closed’ Get3 structure, and this site has been shown to mediate TMD binding 61, 64. In Cy5 Firefly Luciferase mRNA sale to ATP analogs, the Get1 cytosolic domain preferentially binds apo-Get3 in a wide-open conformation (Figure 3A, top left structure) and promotes the release of TA substrate from Get3 58, 65. Kinetic analyses of the Get3 ATPase cycle uncovered additional conformational states. The Get4/5 complex specifically stabilizes ATP binding to Get3 [66]. Consistent with this, crystallographic analyses showed that Get4/5 bridges the dimer interface of ATP-bound Get3 and selectively stabilizes the latter into one of the most closed conformations observed 67, 68. Nevertheless, Get4/5 inhibits ATP hydrolysis by Get3, indicating that Get4/5 induces a distinct ‘occluded’ state of Get3 in which tight and specific ATP binding is uncoupled from ATPase activation (Figure 3A, Step 3) [66]. In contrast to Get4/5, the TA substrate strongly activates the ATPase activity of Get3, indicating that TA-loaded Get3 adopts an ‘activated’ conformation in which its active site is optimized (Figure 3A, Step 4) [66]. Compared with free Get3, TA-loaded Get3 also exhibits significantly weakened affinity for Get4/5 [69], further supporting the notion that Get3 adopts a conformation distinct from the ‘closed’ or ‘occluded’ states upon substrate loading. Finally, while nucleotide-bound Get3–TA complexes exhibit higher affinity for the Get2 than the Get1 subunit (Figure 3A, Step 5), Get1 gains significantly higher affinity for the Get3–TA complex upon ADP release [69]. This suggests another ‘strained’ conformation of the Get3–TA complex (although this could also be explained by a shift in conformational equilibrium of the Get3–TA complex toward the ‘open’ conformation, rather than a distinct conformational state), at which stage Get1 can initiate interaction with and remodeling of the targeting complex (Figure 3A, Step 6). Finally, Get1 exhibits the highest affinity for free apo-Get3, with dissociation rate constants in the low nanomolar range 62, 69; these results, combined with the crystal structures, strongly suggest that a wide-open Get3 bound to Get1 accumulates at the end of the targeting cycle. Together, these results showed that the GET pathway is driven by an ordered series of nucleotide-, substrate-, and effector-driven conformational changes in the Get3 ATPase dimer (Figure 3B). Prior to TA binding, the majority of Get3 in the cytosol is loaded with ATP and tightly bound to Get4/5 (Steps 1 and 2). Get4/5 brings Get3 into the vicinity of Sgt2 (Step 0) and induces Get3 into the optimal conformation and nucleotide state to capture the TA substrate. TA loading drives the dissociation of Get3 from Get4/5 and activates a rapid round of ATP hydrolysis (Step 3). The Get3–TA complex is likely initially captured by the Get2 subunit at the ER membrane (Step 4). ADP release allows the Get3–TA complex to explore additional conformations for which Get1 has higher affinity, and thus initiates the remodeling of the targeting complex (Step 5). The strong preference of Get1 for wide-open Get3 drives the disassembly of the Get3–TA complex (Step 5), followed by insertion of the TA substrate into the ER membrane that may be facilitated by the TMDs of the Get1/2 receptor [60]. At the end of the pathway, Get3 is tightly bound to Get1/2, requiring both ATP and Get4/5 to release it from ER and reinitiate the targeting cycle (Steps 1 and 2) [69].