The protein kinase GCN2 senses the integrity of the multi-tRNA synthetase complex.

Aminoacyl-tRNA synthetases (ARSs) assemble with ARS-interacting multifunctional proteins (AIMPs) to form the multi-tRNA synthetase complex (MSC), a supramolecular complex proposed to enhance efficiency of tRNA aminoacylation. Here, we show that disassembly of MSC via siRNA-mediated depletion of the scaffold protein AIMP2 promotes a transient activation of the eIF2α kinase GCN2 in mouse embryonic fibroblasts (MEFs). Activation of GCN2 was accompanied by increased eIF2α phosphorylation, induction of ATF4, and attenuation of global protein synthesis. Importantly, these effects were abolished in Gcn2-/- MEFs, indicating that translational repression following MSC disassembly is largely dependent on GCN2. Notably, GCN2 activation and eIF2α phosphorylation were induced at early time points and resolved by 24h following AIMP2 depletion and progressively resolved despite sustained suppression of AIMP2 expression, suggesting adaptive restoration of translational homeostasis. Together, our findings link disruption of MSC integrity to the GCN2-dependent integrated stress response and support a functional link between higher-order organization of the translational machinery and cellular stress sensing.