Montero-Lomeli Monica, Morais Bruno L B, Figueiredo Daniela L, Neto Domingos C S, Martins João R P, Masuda Claudio A
Departamento de Bioquimica Médica, ICB-CCS, Universidade Federal do Rio de Janeiro, C.P. 68041, Rio de Janeiro, R.J. 21941-590, Brazil.
J Biol Chem. 2002 Jun 14;277(24):21542-8. doi: 10.1074/jbc.M201977200. Epub 2002 Apr 8.
A gene, TIF2, was identified as corresponding to the translation initiation factor eIF4A and when overexpressed it confers lithium tolerance in galactose medium to Saccharomyces cerevisiae. Incubation of yeast with 6 mm LiCl in galactose medium leads to inhibition of [(35)S]methionine incorporation. By polysome analysis we show that translation is inhibited by lithium at the initiation step, accumulating 80 S monosomes. We further show by immunoblot analysis that when cells are incubated with lithium eIF4A does not sediment with ribosomal subunits. Overexpression of TIF2 overcomes inhibition of protein synthesis and restores its sedimentation with the initiation complex. In vivo, eIF4A is induced by lithium stress. We have shown previously that lithium is highly toxic to yeast when grown in galactose medium mainly due to inhibition of phosphoglucomutase, an enzyme responsible for the entry of galactose into glycolysis. We show that conditions that revert inhibition of phosphoglucomutase also revert inhibition of protein synthesis. Interestingly, glucose starvation leads to loss of polysomes but not to dissociation of eIF4A from the preinitiation complexes. Overexpression of SIT4, a protein phosphatase related to the TOR kinase pathway, reverts inhibition of protein synthesis by lithium and association of eIF4A with the initiation complex.
一个名为TIF2的基因被鉴定为与翻译起始因子eIF4A相对应，当它过表达时，能使酿酒酵母在半乳糖培养基中具有锂耐受性。在半乳糖培养基中用6 mM LiCl孵育酵母会导致[(35)S]甲硫氨酸掺入受到抑制。通过多核糖体分析，我们表明锂在起始步骤抑制翻译，积累80 S单体核糖体。我们通过免疫印迹分析进一步表明，当细胞用锂孵育时，eIF4A不会与核糖体亚基一起沉降。TIF2的过表达克服了蛋白质合成的抑制，并恢复了其与起始复合物的沉降。在体内，eIF4A由锂应激诱导。我们之前已经表明，当在半乳糖培养基中生长时，锂对酵母具有高度毒性，主要是由于抑制了磷酸葡萄糖变位酶，该酶负责半乳糖进入糖酵解。我们表明，恢复磷酸葡萄糖变位酶抑制的条件也能恢复蛋白质合成的抑制。有趣的是，葡萄糖饥饿会导致多核糖体的丢失，但不会导致eIF4A从起始前复合物中解离。与TOR激酶途径相关的蛋白磷酸酶SIT4的过表达可恢复锂对蛋白质合成的抑制以及eIF4A与起始复合物的结合。
