Figure 2
OLA1 binds to eIF2 and interferes with TC formation via its GTPase activity.
(A) Interaction of endogenously expressed OLA1 and eIF2α in HEK293T cells. Cell lysate was immunoprecipitated with anti-eIF2α or anti-IgG antibody and the precipitates were immunoblotted with anti-OLA1 and anti-eIF2α antibodies. (B) Ectopically expressed OLA1 binds with endogenous eIF2α. HEK293T cells were transfected with a control or FLAG-OLA1 vector for 48 h. Cell lysates were subject to IP with anti-FLAG antibody and IB with the indicated antibodies. (C) Ectopically expressed eIF2α binds with endogenous OLA1. HEK293T cells were transfected with a control vector or expression vectors encoding HA-eIF2α or HA-eIF2α-S51A. Cell lysates were subjected to IP with anti-HA antibody and IB with indicated antibodies. (D) The top 10 predicted interactions between OLA1 (blue (#1)→green (#10); PDB ID: 2OHF)16 and aIF2 (red; PDB ID: 3CW2)20 as calculated by the program ZDOCK48. (E) Diagrams of the OLA1-WT and mutant proteins N230A and ΔTGS. (F) In vitro binding of eIF2 with [14C]-labeled yeast initiator Met-tRNAiMet in the presence of GTP. The mean amount of Met-tRNAi bound to 150 nM eIF2 (~0.6 pmol) is set as 100%. (G) The effect of OLA1 proteins on formation of TC. Purified eIF2 was incubated with GTP and OLA1 (or other proteins), followed by addition of [14C]-Met-tRNAiMet. (Protein concentration: 150 nM for each protein.) (H) Nucleotide hydrolysis activity of the OLA1 proteins. Equal amount of protein (WT, N230A, ΔTGS, or actin; 1 μM) was incubated with [γ-32P]GTP or [γ-32P]ATP in reaction buffer at 35 °C for 30 min. The rate of the release of [32P]Pi was used to calculate Kcat. Error bars: SD, Student’s t test: *p < 0.05; **p < 0.01. Cropped blots are used. Full scan images of immunoblots are presented in Figure S11.
