Abstract
The eukaryotic elongation factor 1A (eEF1A) delivers aminoacyl-tRNAs to the ribosomal A-site during protein synthesis. To ensure a continuous supply of amino acids, cells harbor the kinase Gcn2 and its effector protein Gcn1. The ultimate signal for amino acid shortage is uncharged tRNAs. We have proposed a model for sensing starvation, in which Gcn1 and Gcn2 are tethered to the ribosome, and Gcn1 is directly involved in delivering uncharged tRNAs from the A-site to Gcn2 for its subsequent activation. Gcn1 and Gcn2 are large proteins, and these proteins as well as eEF1A access the A-site, leading us to investigate whether there is a functional or physical link between these proteins. Using Saccharomyces cerevisiae cells expressing His(6)-eEF1A and affinity purification, we found that eEF1A co-eluted with Gcn2. Furthermore, Gcn2 co-immunoprecipitated with eEF1A, suggesting that they reside in the same complex. The purified GST-tagged Gcn2 C-terminal domain (CTD) was sufficient for precipitating eEF1A from whole cell extracts generated from gcn2Δ cells, independently of ribosomes. Purified GST-Gcn2-CTD and purified His(6)-eEF1A interacted with each other, and this was largely independent of the Lys residues in Gcn2-CTD known to be required for tRNA binding and ribosome association. Interestingly, Gcn2-eEF1A interaction was diminished in amino acid-starved cells and by uncharged tRNAs in vitro, suggesting that eEF1A functions as a Gcn2 inhibitor. Consistent with this possibility, purified eEF1A reduced the ability of Gcn2 to phosphorylate its substrate, eIF2α, but did not diminish Gcn2 autophosphorylation. These findings implicate eEF1A in the intricate regulation of Gcn2 and amino acid homeostasis.
Highlights
The eukaryotic elongation factor 1A delivers aminoacyl-tRNAs to the ribosomal acceptor site (A-site) during protein synthesis
Gcn2 Co-elutes with His6-eukaryotic elongation factor 1A (eEF1A), but Gcn1 Does Not—As eEF1A accesses the ribosome during the delivery of amino acidtRNAs to the A-site [1], as we have proposed that Gcn1 and Gcn2 detect uncharged tRNAs in the A-site during amino acid starvation [6, 9], and as eEF1A was found to bind uncharged tRNAs [20], we wanted to investigate whether eEF1A is involved in the general amino acid control (GAAC) process
We found that Gcn2 eluted from the iMAC resin only when eEF1A was His6-tagged, and the elution pattern resembled that of His6-eEF1A (Fig. 1), strongly suggesting that Gcn2 and eEF1A reside in the same complex
Summary
GSTb-gcn2(1–272), here designated GST-gcn2-NTD GSTb-gcn2(568–998), here designated GST-gcn2-PK GSTb-gcn2(970–1497), here designated GST-gcn2-HisRS GSTb-gcn2(1498–1659), here designated GST-gcn2-CTD GSTb-gcn2(1498–1659)-K1552L, K1553I, K1556I, here designated. C-terminal to the eIF2␣ kinase domain is a domain with homology to histidyl-tRNA synthetases This HisRS-like domain is enzymatically not functional, and instead it binds uncharged tRNAs, i.e. the amino acid starvation signal. Gcn2-eEF1A interaction is diminished in amino acid-starved cells, and this interaction is disrupted by uncharged tRNAs in vitro, and eEF1A can inhibit the eIF2␣ kinase function of Gcn in vitro. These findings suggest that eEF1A is a negative effector of the GAAC response in amino acid-replete cells, adding a new player to the complex regulatory network that couples rates of protein synthesis and amino acid production to nutrient availability
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