Functional analysis of individual binding activities of the scaffold protein eIF4G.
Eukaryotic initiation factor (eIF) 4G is an integral member of the translation initiation machinery. The molecule serves as a scaffold for several other initiation factors, including eIF4E, eIF4AI, the eIF3 complex, and poly(A)-binding protein (PABP). Previous work indicates that complexes between these proteins exhibit enhanced mRNA cap-binding and RNA helicase activities relative to the respective individual proteins, eIF4E and eIF4A. The eIF4G-PABP interaction has been implicated in enhancing the formation of 48 S and 80 S initiation complexes and ribosome recycling through mRNA circularization. The eIF3-eIF4GI interaction is believed to forge the link between the 40 S subunit and the mRNA. Here we have investigated the behavior in vitro and in intact cells of eIF4GIf molecules lacking either the PABP-binding site, the eIF3-binding site, the middle domain eIF4A-binding site, or the C-terminal segment that includes the second eIF4A-binding site. Although in some cases the mutant forms were recruited more slowly, all of these eIF4G variants could form complexes with eIF4E, enter 48 S complexes and polysomes in vivo and in vitro, and partially rescue translation in cells targeted with eIF4GI short interfering RNA. In the reticulocyte lysate, eIF4G unable to interact directly with PABP showed little impairment in its ability to support translation, whereas loss of either of the eIF4A-binding sites or the eIF3-binding site resulted in a marked decrease in activity. We conclude that there is considerable redundancy in the mechanisms forming initiation complexes in mammalian cells, such that many individual interactions have regulatory rather than essential roles.
Hinton
Tracey M
TM
Department of Biochemistry, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.
Coldwell
Mark J
MJ
Carpenter
Gillian A
GA
Morley
Simon J
SJ
Pain
Virginia M
VM
D000818:Animals; D001665:Binding Sites; D039603:Eukaryotic Initiation Factor-4G / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007313:Insecta; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D010447:Peptide Hydrolases / Q000737:chemistry; D039102:Poly(A)-Binding Proteins / Q000378:metabolism; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D039603:Eukaryotic Initiation Factor-4G; D039102:Poly(A)-Binding Proteins; D034741:RNA, Small Interfering; D010447:Peptide Hydrolases