eIF2β zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding

Abstract The heterotrimeric eIF2 complex consists of a core eIF2γ subunit to which binds eIF2α and eIF2β subunits and plays an important role in delivering the Met-tRNAiMet to the 40S ribosome and start codon selection. The intricacies of eIF2β-γ interaction in promoting Met-tRNAiMet binding are not clearly understood. Previously, the zinc-binding domain (ZBD) eIF2βS264Y mutation was reported to cause Met-tRNAiMet binding defect due to the intrinsic GTPase activity. We showed that the eIF2βS264Y mutation has eIF2β-γ interaction defect. Consistently, the eIF2βT238A intragenic suppressor mutation restored the eIF2β-γ and Met-tRNAiMet binding. The eIF2β-ZBD residues Asn252Asp and Arg253Ala mutation caused Met-tRNAiMet binding defect that was partially rescued by the eIF2βT238A mutation, suggesting the eIF2β-ZBD modulates Met-tRNAiMet binding. The suppressor mutation rescued the translation initiation fidelity defect of the eIF2γN135D SW-I mutation and eIF2βF217A/Q221A double mutation in the HTH domain. The eIF2βT238A suppressor mutation could not rescue the eIF2β binding defect of the eIF2γV281K mutation; however, combining the eIF2βS264Y mutation with the eIF2γV281K mutation was lethal. In addition to the previously known interaction of eIF2β with the eIF2γ subunit via its α1-helix, the eIF2β-ZBD also interacts with the eIF2γ subunit via guanine nucleotide-binding interface; thus, the eIF2β-γ interacts via two distinct binding sites.


Screening of the eIF2b S264Y intragenic suppressor muta5on
A single copy plasmid carrying the eIF2b S264Y mutant gene was subjected to random mutagenesis using XL-1 Red E. coli mutagenic strain to prepare a library of mutant plasmids.We screened the library of mutant plasmids to identify mutations that suppressed the slow-growth phenotype associated with the eIF2b S264Y mutation.Out of 1000 colonies screened, 7 colonies showed better growth phenotype than the original eIF2b S264Y mutation.Sequencing of the plasmids isolated from these 7 colonies revealed two revertant mutations, one mutation converted the original eIF2b S264Y residue to alanine, and four plasmids showed eIF2b-Thr238 to Ala change.Yeast strain YP896 (his4D, sui3Δ) carrying single copy YCplac111_HA_SUI3 (A202) or YCplac111_HA_SUI3-T238A (A1394) plasmids were grown overnight.The whole cell extract (WCE) was prepared by mechanical cell breaking using glass beads.The N-terminal end of the eIF2b protein was fused with a 3xHA tag, and using anti-HA antibody agarose beads, the eIF2b protein was coimmunoprecipitated from the WCE and subjected to the Western blotting using an anti-HA antibody (eIF2b), anti-eIF2g, anti-eIF2a, anti-eIF2Be , and RPS20 antibodies.Half of the Co-IP beads were treated with Trizol, and the RNA was precipitated with ethanol and subjected to the Northern blot analysis using a probe specific to the initiator Met-tRNAi.Full membrane images of Western and Northern blot showing red highlighted region (bottom).

Figure S2. Analysis of TC on the 43-48S ribosome.
Yeast strain YP912 (sui3D, gcd11D) carrying derivatives of eIF2b mutant were grown overnight A600 ~0.8.The WCE (A260 ~20 U) was layered on a 15%-40% sucrose gradient and resolved by ultracentrifugation at 39,000 rpm (Beckman SW41 rotor) for 5 hrs at 4°C.A 0.7 ml of 15 factions was collected by continuously monitoring A260 nm (Polysome profile: top panel: A).Part of these factions was resolved on the 12% SDS-PAGE (middle panel: B) and analyzed by the Western blotting using indicated antibodies or by Northern blot to identify Met-tRNAi (panel C), Fraction #6-9 (dotted region) were re-ran on separate gels and analyzed by Western or Northern blot to identify 40S subunit fractions (panel: D), Fractions #7 and #8 were identified to contain the majority of 40S-48S complexes.Using these parameters, the yeast strain YP912 carrying derivatives of eIF2b mutant were subjected to 1% HCHO cross-linking as described in Materials and Methods and fractionated as described above.The fractions #1-12 were analysed by Western blot using indicated antibodies or Northern blot to identify Met-tRNAi.The fractions #7 and #8 containing 40S-48S complexes is marked with the dotted lines that shows the corresponding ribosome profile at the bottom.In; is input.Full membrane images (Western and Northern blot showing red highlighted region) of TC on the 43-48S ribosome (bottom or right side).A) Growth analysis.Yeast strain YP896 (his4D, sui3Δ) carrying single copy eIF2b WT (A1451), eIF2b F217A,Q221A (A1490), eIF2b F217A,Q221A/T238A (A1492) or eIF2b T238A (A1260) plasmids were grown overnight, serially diluted, and spotted on SD plus uracil, tryptophan, and histidine plate and incubated at 30°C for 2-3 days.B) Analysis of HIS4-lacZ expression.Yeast strains from (A) were transformed with either A1072 (GAPDHprom_His4 AUG _lacZ) or A1073 (GAPDHprom_His4 UUG _lacZ) plasmid constructs and grown on the SCD plus tryptophan media and harvested at OD600 ~ 0.8.The harvested cells were subjected to b-galactosidase assay (nmol of O-nitrophenyl-b-Dgalactopyranoside cleaved per min per mg), and the resultant values were plotted as UUG/AUG ratio.C) Analysis of GCN4-lacZ expression.Yeast strains from (A) were transformed with GCN4-lacZ construct (p180).The measurement of β-galactosidase activity was done as described above.
Statistical differences were determined by one-way ANOVA analysis.The error bar shows the standard deviation.

Figure S3 .
Figure S3.Analysis of the purified WT and mutant eIF2 complex.The eIF2 complexes were purified from strains overexpressing eIF2abg subunits as described in Materials and Methods.Different amounts (0.5 and 2 µg) of the eIF2 complexes were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Western blot (top) or Coomassie blue stain (bottom).The positions of the eIF2g, eIF2a , and eIF2b subunits are shown on the right.Full membrane images of Western blot showing red highlighted region (bottom).

Figure S4 .
Figure S4.The eIF2b T238A mutation suppresses the Sui¯ and Gcd¯ phenotype of the eIF2b HTH mutants.