Critical Contacts between the Eukaryotic Initiation Factor 2B (eIF2B) Catalytic Domain and both eIF2β and -2γ Mediate Guanine Nucleotide Exchange

Autor: Sarah S. Mohammad-Qureshi, Raphaël Haddad, Jonathan P. Richardson, Graham D. Pavitt, Elizabeth J. Hemingway
Rok vydání: 2007
Předmět:
Zdroj: Molecular and Cellular Biology. 27:5225-5234
ISSN: 1098-5549
DOI: 10.1128/mcb.00495-07
Popis: The cap-dependent pathway for the initiation of translation requires the assembly of eukaryotic initiation factor (eIF) ribosomal subunits and a selected mRNA. Central to the initiation process is the GTP binding protein eIF2, which delivers aminoacylated initiator methionyl tRNA () to the 40S ribosome as part of a multifactor complex containing eIFs 1, 3, and 5 (5). This 43S preinitiation complex associates with the 5′ end of an mRNA and migrates along it to locate an AUG initiator codon with the aid of other factors. AUG codon recognition, eIF5-promoted hydrolysis of GTP bound to eIF2, and phosphate release stimulate eIF2-GDP and eIF5 dissociation from the initiation complex, probably as an eIF2-GDP/eIF5 complex (33). remains bound to the 40S ribosomal subunit at the AUG and is probably stabilized by eIF1A/eIF5B-GTP. 60S ribosomal subunit joining is accelerated by GTP hydrolysis and the release of eIF5B-GDP. At this point, translation elongation can commence (20). The regeneration of eIF2-GTP from the inactive GDP-bound complex released from the initiation complex is carried out by the guanine nucleotide exchange factor (GEF) eIF2B. eIF2B accelerates the otherwise slow dissociation of GDP from eIF2, allowing its replacement with GTP. This process is regulated by the phosphorylation of eIF2α on the conserved ser51 residue in response to a diverse set of cellular stresses (20). For example, in yeast cells (Saccharomyces cerevisiae), translational control of GCN4 expression proceeds by the following pathway: amino acid starvation activates Gcn2p, which phosphorylates eIF2α and inhibits eIF2B activity. This lowers the rate of nucleotide exchange and slows recruitment of the eIF2-GTP/ternary complex (TC) to mRNAs. GCN4 translation is activated by this response, as it contains short upstream open reading frames that normally limit the flow of scanning ribosomes to the GCN4 initiator codon and repress its translation. Starvation for one or more amino acids affects reinitiation within the GCN4 5′ leader such that inhibitory upstream open reading frames are bypassed and GCN4 translation is enhanced by up to 10-fold (23). Inherited eIF2B mutations are responsible for a fatal human disorder known as childhood ataxia with central nervous system hypomyelination or leukoencephalopathy with vanishing white matter (18). This chronic progressive disease results in the aberrant development or destruction of glial cells within the brain. Cellular disease models show that eIF2B mutations lower the nucleotide exchange activity of eIF2B and can elevate cellular stress responses in both nonglial cells (19, 25, 31) and glia (39) and impair the development of astrocytes (14). As would be expected for a general protein synthesis factor defect, mutations that are associated with more severe disease pathologies affect other tissues and result in early mortality (38). Disease-causing mutations have been found in all eIF2B subunits. eIF2B is a heteropentameric complex, consisting of subunits eIF2Bα to eIF2Bɛ, encoded by five different genes. The eIF2Bα, -β, and -δ subunits form the “regulatory” subcomplex that down-regulates eIF2B activity in response to the phosphorylation of ser51 on eIF2α. The phosphorylated form of eIF2 is more tightly bound by the regulatory subcomplex but prevents the exchange of guanine nucleotide, resulting in a reduction in the availability of eIF2B to regenerate active eIF2 (28). The eIF2Bγ and eIF2Bɛ subunits form the “catalytic” subcomplex that is required for accelerating the rate of guanine nucleotide exchange. eIF2Bγ and eIF2Bɛ share significant sequence homology with each other and possess motifs also present in other protein families, including a large family of nucleotidyl transferases (pfam00483) and a hexapeptide repeat common in acyltransferases (pfam00132) (3). The functional significance of these sequence similarities is not clear. In addition, the C-terminal domain (CTD) of eIF2Bɛ is homologous both to the eIF5-CTD and to eIF4G (pfam02020) (8, 9, 40). It is this last domain that contains residues sufficient for GEF activity of eIF2B, albeit at a reduced rate compared with that of the full five-subunit complex (21). The domain consists of stacked pairs of α-helices and has been called a modified HEAT repeat. To characterize residues within this catalytic domain (ɛcat) that are essential for nucleotide exchange, we have undertaken a site-directed mutagenesis (SDM) approach, targeting conserved surface residues, followed by extensive genetic and biochemical studies. We find that at least two residues on opposing sides of the domain are critical for the nucleotide exchange function by eIF2B, E569 and W699, as mutations of these residues are lethal in vivo. Nonlethal mutations within residues on the same surface as E569 are cold sensitive for growth and GCN4 activity and salt sensitive for eIF2 interaction. By examining interactions between individual eIF2 subunits, we show that W699 is critical for interaction with eIF2β, while L568 and E569 mutations do not significantly alter this interaction. Instead, mutations to all three residues are impaired for eIF2γ binding. The combined genetic and biochemical data support the ideas that interactions between ɛcat and eIF2γ are extensive and that contacts between ɛcat and both eIF2β and eIF2γ contribute to eIF2B nucleotide exchange function.
Databáze: OpenAIRE
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