A novel mechanism for the control of translation initiation by amino acids, mediated by phosphorylation of eukaryotic initiation factor 2B.

Eukaryotic initiation factor 2B (eIF2B) plays a key role in controlling the initiation of mRNA translation. eIF2B is heteropentamer whose catalytic (epsilon) subunit promotes GDP/GTP exchange on eIF2. We show here that depriving human cells of amino acids rapidly results in the inhibition of eIF2B, independently of changes in eIF2 phosphorylation. Although amino acid deprivation also inhibits signaling through the mammalian target of rapamycin complex 1 (mTORC1), the inhibition of eIF2B activity by amino acid starvation is independent of mTORC1. Instead, amino acids repress the phosphorylation of a novel site in eIF2Bepsilon. We identify this site as Ser525, located adjacent to the known phosphoregulatory region in eIF2Bepsilon. Mutation of Ser525 to Ala abolishes the regulation of eIF2B and protein synthesis by amino acids. This indicates that phosphorylation of this site is crucial for the control of eIF2B and protein synthesis by amino acids. These findings identify a new way in which amino acids regulate a key step in translation initiation and indicate that this involves a novel amino acid-sensitive signaling mechanism.

Vanishing white matter disease in a child presenting with ataxia.

Vanishing white matter disease is a recently described leukoencephalopathy that is characterized by chronic and episodic neurological deterioration. These episodes often follow periods of fever or minor head trauma. It frequently presents in childhood with problems of ataxia and tremor. Five genes have been identified for the disease, EIF2B1-5, which encode the five subunits of translation initiation factor eIF2B. Mutations in each of the genes may independently cause the disease. The defect in eIF2B results in abnormalities in translation and its regulation, leading to abnormalities in protein synthesis and its regulation. Magnetic resonance imaging of the brain reveals extensive cerebral white matter abnormalities with evidence of white matter rarefaction and cystic degeneration, which has been confirmed pathologically. We report the first confirmed Australasian patient.

Purification and kinetic analysis of eIF2B from Saccharomyces cerevisiae.

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine nucleotide exchange factor for translation initiation factor 2 (eIF2). Recent studies in the yeast Saccharomyces cerevisiae have served to characterize genetically the exchange factor. However, enzyme kinetic studies of the yeast enzyme have been hindered by the lack of sufficient quantities of protein suitable for biochemical analysis. We have purified yeast eIF2B and characterized its catalytic properties in vitro. Values for K(m) and V(max) were determined to be 12.2 nm and 250.7 fmol/min, respectively, at 0 degrees C. The calculated turnover number (K(cat)) of 43.2 pmol of GDP released per min/pmol of eIF2B at 30 degrees C is approximately 1 order of magnitude lower than values previously reported for the mammalian factor. Reciprocal plots at varying fixed concentrations of the second substrate were linear and intersected to the left of the y axis. This is consistent with a sequential catalytic mechanism and argues against a ping-pong mechanism similar to that proposed for EF-Tu/EF-Ts. In support of this model, our yeast eIF2B preparations bind guanine nucleotides, with an apparent dissociation constant for GTP in the low micromolar range.