Vornlocher H P, Hanachi P, Ribeiro S, Hershey J W
Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616, USA.
J Biol Chem. 1999 Jun 11;274(24):16802-12. doi: 10.1074/jbc.274.24.16802.
Translation initiation factor eIF3 is a multisubunit protein complex required for initiation of protein biosynthesis in eukaryotic cells. The complex promotes ribosome dissociation, the binding of the initiator methionyl-tRNA to the 40 S ribosomal subunit, and mRNA recruitment to the ribosome. In the yeast Saccharomyces cerevisiae eIF3 comprises up to 8 subunits. Using partial peptide sequences generated from proteins in purified eIF3, we cloned the TIF31 and TIF32 genes encoding 135- (p135) and 110-kDa (p110) proteins. Deletion/disruption of TIF31 results in no change in growth rate, whereas deletion of TIF32 is lethal. Depletion of p110 causes a severe reduction in cell growth and protein synthesis rates as well as runoff of ribosomes from polysomes, indicative of inhibition of the initiation phase. In addition, p110 depletion leads to p90 co-depletion, whereas other eIF3 subunit levels are not affected. Immunoprecipitation or nickel affinity chromatography from strains expressing (His)6-tagged p110 or p33 results in the co-purification of the well characterized p39 and p90 subunits of eIF3 as well as p110 and p33. This establishes p110 as an authentic subunit of eIF3. In similar experiments, p135 and other eIF3 subunits sometimes, but not always, co-purify, making assignment of p135 as an eIF3 subunit uncertain. Far Western blotting and two-hybrid analyses detect a direct interaction of p110 with p90, p135 with p33, and p33 with eIF4B. Our results, together with those from other laboratories, complete the cloning and characterization of all of the yeast eIF3 subunits.
翻译起始因子eIF3是真核细胞中蛋白质生物合成起始所需的多亚基蛋白质复合物。该复合物促进核糖体解离、起始甲硫氨酰 - tRNA与40 S核糖体亚基的结合以及mRNA募集到核糖体。在酿酒酵母中，eIF3由多达8个亚基组成。利用从纯化的eIF3中的蛋白质产生的部分肽序列，我们克隆了编码135 kDa（p135）和110 kDa（p110）蛋白质的TIF31和TIF32基因。TIF31的缺失/破坏导致生长速率无变化，而TIF32的缺失是致死的。p110的缺失导致细胞生长和蛋白质合成速率严重降低以及核糖体从多聚核糖体上脱落，这表明起始阶段受到抑制。此外，p110的缺失导致p90共同缺失，而其他eIF3亚基水平不受影响。从表达（His）6标记的p110或p33的菌株中进行免疫沉淀或镍亲和层析，结果显示eIF3中已明确特征的p39和p90亚基以及p110和p33共同纯化。这确定了p110是eIF3的一个真实亚基。在类似实验中，p135和其他eIF3亚基有时（但并非总是）共同纯化，这使得将p135确定为eIF3亚基存在不确定性。Far Western印迹法和双杂交分析检测到p110与p90、p135与p33以及p33与eIF4B之间的直接相互作用。我们的结果与其他实验室的结果一起，完成了所有酵母eIF3亚基的克隆和表征。
