Chapter 5 eEF1A interacts with protein kinase Gcn2
5.5 Direct interaction between eEF1A and Gcn2
The next approach used to ensure that the interaction is not bridged by any other molecule in the yeast whole cell extract was an in vitro binding assay with purified eEF1A. Purified eEF1A was supplied by Dr. Sattlegger. Briefly a GCN2Δyeast strain with His6-eEF1A as the only form of eEF1A (ESY10101) was grown and the whole cell extract was prepared. The whole cell extract was then incubated with Ni charged
96 resin. The resin was subsequently washed to remove all unbound proteins. The His6- eEF1A was then eluted with buffer containing 250mM imidazole. The eluate was resolved by SDS PAGE. The gel was stained using Coomassie brilliant blue stain to check for any contaminating proteins in the purified eEF1A protein; none were detectable (Figure 5.7).
Figure 5.7: Coomassie stained gel with purified eEF1A
His6-eEF1A was purified from WCE of ESY10101 by affinity binding to a Ni charged resin and eluted with 250mM immidazole. The purified His6-eEF1A was resolved on a 10% polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue stain for 30 min and destained until bands were clearly visible.
The GST-Gcn2-CTD fragment, GST-Gcn2-CTD K3 and GST alone were immobilised on glutathione beads followed by incubation with purified His6-eEF1A. The precipitates were resolved by SDS PAGE and immunoblotting with antibodies against eEF1A and GST. eEF1A associated with both Gcn2-CTD and Gcn2-CTD K3 (lanes 2 and 3 Figure 5.8). However, Gcn2-CTD K3 precipitated less eEF1A in comparison to the Gcn2-CTD fragment. This provides further proof that the interaction between Gcn2 and eEF1A is not bridged by the ribosome or other proteins from the yeast extract.
97 Figure 5.8: Direct interaction between eEF1A and Gcn2
GST tagged Gcn2-CTD and Gcn2-CTD K3 fragments, GST alone expressed and purified from E. coli were immobilized on glutathione beads and incubated with purified eEF1A. The proteins bound to the beads were then resolved on a SDS- polyacrylamide gel and subjected to immunodetection using antibodies against eEF1A and GST.
It is worth noting that the lysine substitutions mentioned in section 5.4 affected
Gcn2 CTD’s ability to bind eEF1A. Not only does Gcn2-CTD K3 bind eEF1A weaker than Gcn2-CTD (compare lanes 2 and 3 Figure 5.8), it also required about twice as much of Gcn2 CTD K3 when compared to Gcn2 CTD (compare lanes 3 with 2 bottom panel Figure 5.8). When the signal intensities were quantified and normalised for GST-Gcn2, it was found that the Gcn2-CTD K3 bound 60% less eEF1A in comparison to Gcn2-CTD (Figure 5.9). This indicates that the lysine substitutions in Gcn2-CTD reduced affinity for eEF1A.
Figure 5.9: Amount of eEF1A bound to the Gcn2 fragments
The amount of eEF1A bound to GST-Gcn2 CTD or GST-Gcn2 CTD K3 was quantified using Multi Gauge V3.1 software (Fuji Photo Film Co., Ltd.) and plotted normalised to GST.
0 0.5 1 1.5 2 GST-Gcn2 CTD GST-Gcn2 CTD K3 eE F1A bound n o rm al is ed to G ST
98 Since eEF1A and Gcn2 are both known to bind tRNAs (Dong et al., 2000; Legocki et al., 1974), their interaction could possibly be mediated by RNA molecules. To ensure that the interaction was not bridged by RNA molecules, the in vitro binding assay carried out in Figure 5.8 was repeated with purified eEF1A and the Gcn2 fragments that were pre-treated with RNase A for 15 minutes on ice, as published previously (Marton et al., 1997). The efficiency of the RNase treatment was confirmed by digesting 1μg of yeast RNA at conditions identical to those in the assay. The treated and untreated RNA samples were resolved on a 1% agarose gel. No RNA could be detected after the RNase treatment (compare treated and untreated lanes Figure 5.10 A), indicating that the RNase treatment was efficient and complete.
Figure 5.10: eEF1A-Gcn2 interaction is not bridged by RNA
(A) 1μg of yeast total RNA was digested with RNase A for 15 min on ice. The untreated control and treated samples were run on an agarose gel. The gel was stained with ethidium bromide and visualised under UV light. (B) RNase treated and untreated GST alone, GST tagged Gcn2-CTD and Gcn2-CTD K3 fragments that were expressed and purified from E. coli were immobilized on glutathione beads and incubated with RNase treated and untreated purified eEF1A. The proteins bound to the beads were then resolved in a SDS-polyacrylamide gel and subjected to immunodetection using antibodies against eEF1A and GST.
The RNase-digested GST-Gcn2-CTD fragment, Gcn2-CTD K3 and GST alone were immobilised on glutathione beads, followed by incubation with RNase digested purified eEF1A. The precipitates were resolved by SDS PAGE and analysed by immunoblotting with antibodies against eEF1A and GST. Similar amounts of eEF1A associated with Gcn2-CTD and Gcn2-CTD K3 under both RNase treated and untreated conditions (Figure 5.10 B, compare lanes 3 and 4, lanes 6 and 7). The
99 signal intensities of eEF1A precipitated by GST-Gcn2 CTD and GST-Gcn2 CTDK3 were quantified and normalised against GST-Gcn2 (CTD or CTDK3) and plotted in a graph (Figure 5.11). The RNase treatment did not seem to affect the amount of eEF1A bound by the Gcn2-CTD and Gcn2-CTD K3 fragments (compare lanes 4 and 7, lanes 3 and 6, Figure 5.11). This indicates that the Gcn2 and eEF1A interaction is not mediated by RNA molecules. Once again, the lysine mutations (K3) were shown to affect Gcn2’s ability to bind eEF1A. It required twice as much of the Gcn2-CTD K3 to bind the same amount of eEF1A when compared to Gcn2-CTD (Figure 5.11).
Figure 5.11: Similar amounts of eEF1A bound the Gcn2 fragments with or without RNase digestion
The amount of eEF1A bound to GST-Gcn2 CTD, GST-Gcn2 CTD K3 and GST from Figure 5.10 was quantified using Multi Gauge V3.1 software (Fuji Photo Film Co., Ltd.) in both treated and untreated samples. The amount of eEF1A bound was normalised to GST-Gcn2 and plotted.
With the above observations taken together, it can be concluded that the interaction between Gcn2 and eEF1A is not bridged by the ribosome, RNA or other proteins from the yeast extract.