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. 1999 Apr 13;96(8):4342–4347. doi: 10.1073/pnas.96.8.4342

Figure 2.

Figure 2

hIF2 substitutes for yIF2 to stimulate translation. (A) Protein immunoblot analysis of hIF2. (Left) Twenty micrograms of crude HeLa cell extract was subjected to SDS/PAGE and then blotted to a nitrocellulose membrane. The blot was probed with affinity-purified anti-hIF2 antibodies that were raised against a MBP-hIF2 fusion protein containing the C-terminal half of hIF2 (lane 1), then the blot was stripped and probed with antiserum raised against an N-terminal hIF2 peptide (lane 2). (Right, lanes 3–6) 20 μg of crude yeast cell extracts from a fun12Δ strain expressing the indicated hIF2 proteins was subjected to SDS/PAGE and then blotted to a nitrocellulose membrane. The blot was probed with the affinity-purified anti-hIF2 antibodies, and then stripped and probed with anti-eIF2α antiserum, as indicated. Positions of protein molecular mass markers (kDa) are indicated on the left. (B) Expression of hIF2 in yeast complements the slow-growth phenotype of a fun12Δ strain. The fun12Δ strain J133 [Mataura3–52 leu2–3 leu2–112 fun12Δ] was transformed with the vector pEMBLyex4 (vector), the pEMBLyex4-hIF2 expression plasmids pC602 (hIF2), pC612 (hIF2-V640G), or pC613 (hIF2-H706E), or the pEGKT-GST-yIF2 plasmid pC485 (yIF2) (9). The indicated strains were streaked on synthetic minimal medium containing 10% galactose plus the required nutrient supplements, and the plates were incubated at 30°C for 5 days. (C) Restoration of translational activity in extracts from fun12Δ strains by addition of purified hIF2. In vitro translation extracts were prepared from the fun12Δ strain J133 carrying the low-copy-number plasmid pC479 containing wild-type FUN12 (FUN12+) or the vector pRS316 only (fun12Δ). Extracts were incubated with 200 ng of luciferase mRNA and the indicated amounts of recombinant GST, GST-yIF2, or the indicated GST-hIF2 wild-type or mutant fusion proteins. Translational activity was determined by measuring luminescence after 15-min incubation at 26°C. The results presented are representative of at least three independent experiments.