Synthesis of human initiation factor-2 alpha in Saccharomyces cerevisiae.
A human eIF-2 alpha cDNA (encoding alpha-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-2 alpha with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2 alpha 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-2 alpha, suggesting covalent modification in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2 alpha 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 (alpha, beta and gamma) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2 alpha 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-2 alpha subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.
Green
S R
SR
Biological Laboratory, University of Kent, Canterbury, U.K.
Spalding
A
A
Ashford
T
T
Proud
C G
CG
Tuite
M F
MF
D015852:Eukaryotic Initiation Factor-2 / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005690:Galactose / Q000494:pharmacology; D015966:Gene Expression Regulation, Fungal; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic / Q000235:genetics
D015852:Eukaryotic Initiation Factor-2; D011994:Recombinant Proteins; D005690:Galactose