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Molecular and Cellular Biology, July 2007, p. 5225-5234, Vol. 27, No. 14
0270-7306/07/$08.00+0     doi:10.1128/MCB.00495-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Critical Contacts between the Eukaryotic Initiation Factor 2B (eIF2B) Catalytic Domain and both eIF2ß and -2{gamma} Mediate Guanine Nucleotide Exchange{triangledown},{ddagger}

Sarah S. Mohammad-Qureshi, Raphaël Haddad, Elizabeth J. Hemingway, Jonathan P. Richardson,{dagger} and Graham D. Pavitt*

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

Received 21 March 2007/ Returned for modification 18 April 2007/ Accepted 9 May 2007

Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (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{varepsilon} 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 discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15°C. The 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 eIF2ß, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2{gamma}. These data show that multiple contacts between eIF2{gamma} and eIF2B{varepsilon} mediate nucleotide exchange.

* Corresponding author. Mailing address: Faculty of Life Sciences, The University of Manchester, Manchester M13 9PT, United Kingdom. Phone: 44 161 306 4477. Fax: 44 161 236 0409. E-mail: graham.pavitt{at}manchester.ac.uk

{ddagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{triangledown} Published ahead of print on 25 May 2007.

{dagger} Present address: Department of Chemistry, Christopher Ingold Laboratories, University College London, 20 Gordon Street, London WC1H 0AJ, United Kingdom.

Molecular and Cellular Biology, July 2007, p. 5225-5234, Vol. 27, No. 14
0270-7306/07/$08.00+0     doi:10.1128/MCB.00495-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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