Purification and characterization of eukaryotic polypeptide chain initiation factor 4F from Drosophila melanogaster embryos.
The eukaryotic polypeptide chain initiation factor 4F (eIF-4F), purified by m7GTP-Sepharose chromatography from whole extracts of Drosophila melanogaster embryos, consists of two subunits, the previously identified eIF-4E (35 kDa) (Maroto, F. G., and Sierra, J. M. (1989) Mol. Cell. Biol. 9, 2181-2190) and another of 200 kDa. Both subunits cosedimented through a sucrose density gradient containing 0.5 M KCl. In contrast to rabbit reticulocyte eIF-4F, we did not find any RNA-dependent ATPase associated with the Drosophila factor. As shown previously for eIF-4E, the p200 subunit was also required for the translation of endogenous mRNAs in cell-free systems from Drosophila embryos. Only the eIF-4E subunit was able to cross-link to the m7G cap structure. However, an efficient cross-linking of the p200 subunit to an uncapped mRNA was observed. Both subunits were phosphorylated in vitro by protein kinase C from rat brain. As an extension of our previous results (Zapata, J. M., Maroto, F. G., and Sierra, J. M. (1991) J. Biol. Chem. 266, 16007-16014) we found that the translation of the heat shock mRNAs was independent of both of the eIF-4F subunits.
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
J M
JM
Zapata
M A
MA
Martínez
J M
JM
Sierra
D000818:Animals; D002850:Chromatography, Gel; D004331:Drosophila melanogaster / Q000196:embryology; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D011817:Rabbits; D013696:Temperature
D010446:Peptide Fragments; D010448:Peptide Initiation Factors; C075833:eIF-4; C069883:initiation factor 4F component p220