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Mol. Cell. Biol. doi:10.1128/MCB.00495-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Critical contacts between the eIF2B Catalytic Domain and both eIF2 and Mediate Guanine Nucleotide Exchange

Faculty of Life Sciences, The University of Manchester, Manchester, M13 9PT, United Kingdom

^* To whom correspondence should be addressed. Email: graham.pavitt{at}manchester.ac.uk.

	Abstract

Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP-binding proteins. One of the most complicated pairs is eIF2B and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2B C-terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity, and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernable effect on growth or GCN4 expression, but alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15°C. Mutation of W699, found on a separate surface approximately 40 Å from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2 while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2. These data show that multiple contacts between eIF2 and eIF2B mediate nucleotide exchange.