Importantly each conformational state in the SRP

Importantly, each conformational state in the SRP–SR dimer provides a distinct point of regulation at which these GTPases can directly sense and respond to different biological cues in the pathway. For example, assembly of a stable ‘closed’ complex between SRP and SR is intrinsically very slow (kon ∼102–103 M–1 s–1) 32, 35, 45, primarily due to the labile nature of the ‘early’ intermediate, over 98% of which dissociates before it rearranges into the stable ‘closed’ complex [37]. However, RNCs bearing SRP substrates stabilize the ‘early’ intermediate over 100-fold and thus accelerate assembly of the ‘closed’ complex up to 103-fold (Figure 2A, Steps 1 and 2) 46, 47, 48. Rearrangement to the ‘closed’ state is further driven by the interaction of SR with anionic phospholipids (Figure 2A, Step 3) [49], allowing a stable RNC–SRP–SR ‘closed’ targeting complex to accumulate at the membrane. Finally, the last rearrangement that leads to GTPase activation is strongly inhibited by the RNC 47, 50; this effect, termed ‘pausing’, is reversed when the targeting complex contacts the SecYEG translocon (Figure 2A, Step 4) 50, 51. ‘Pausing’ serves two roles: (i) as a ‘timer’ that gives the targeting complex an extended time window to search for the SecYEG complex, minimizing premature GTP hydrolysis, which would lead to abortive pathways; and (ii) as a spatial sensor that couples GTP hydrolysis to the successful unloading of cargo at the membrane translocon. Collectively, these findings provide a high-resolution model for how the GTPase A-674563 in the SRP–SR dimer provides spatiotemporal coordination of co-translational protein targeting (Figure 2B). SRP–SR interaction is minimized in the absence of cargo and is initiated only when SRP binds RNCs bearing SRP substrates (Steps 1 and 2). Before engaging the membrane translocon, the RNC–SRP–SR complex is primarily in the ‘early’ conformational state in which the RNC is tightly bound to SRP, and GTP hydrolysis is delayed. Interactions of SR with phospholipids and with the SecYEG translocon induce GTPase rearrangements into the ‘closed’ and ‘activated’ states, in which the SRP–SR NG-domain complex detaches from the ribosome exit site and moves to the distal site of the SRP RNA (Steps 3 and 4). These rearrangements reduce the affinity of the SRP–SR complex for RNC over 30-fold and free up the ribosome exit site for subsequent docking onto the SecYEG translocon, initiating a sequential and coordinated cargo handover event [52] (Step 5). The same rearrangement also activates GTP hydrolysis in the SRP–SR complex, driving its disassembly and recycling (Step 5). Thus, each conformational change in the SRP–SR GTPase dimer allows it to communicate with the cargo, membrane, and translocon; these allosteric communications provide the driving force for the targeting pathway and have also been shown to generate fidelity checkpoints to enhance the accuracy of substrate selection by SRP 36, 48, 53.
The Get3 Homodimer: ATPase Tangos Drive Post-Translational Protein Targeting Get3 (or TRC40 in mammalian cells) belongs to the ArsA subfamily of ATPases, represented by the arsenic-translocating ATPase ArsA (Figure 1A). Recent biochemical and genetic analyses showed that in eukaryotic cells, Get3 (and TRC40) mediates the delivery of an essential class of membrane proteins, termed ‘tail-anchored (TA) proteins’ as their sole TMD resides near the C terminus, to the ER 54, 55, 56, 57. This process, termed the guided-entry-of-tail-anchored proteins (GET) pathway, begins with the co-chaperone Sgt2 that captures TA proteins released from the ribosome [56]. TA proteins are then transferred from Sgt2 to Get3, bridged by a scaffolding complex consisting of Get4 and Get5 subunits [56]. A receptor complex on the ER membrane, comprising Get1 and Get2 subunits, captures the Get3–TA complex and drives the dissociation of TA proteins from Get3 and its insertion into the membrane 57, 58, 59, 60.