Synthesis of human initiation factor-2α in Saccharomyces cerevisiae

Abstract

A human eIF-2α, cDNA (encoding α-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GAL1.10 promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2α with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2α subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2α, suggesting covalent modifi cation in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2α protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl. This possible association of the synthesised human subunit into a three-subunit (α, β and γ) eIF-2 complex was further examined by partial purifi cation of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2α was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer. These analyses suggest that human eIF-2α subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.