Department of Microbiology and Immunology, University of California, 513 Parnassus Avenue, Room S-472, San Francisco, CA 94143, USA.
Proc Natl Acad Sci U S A. 2011 Apr 19;108(16):6415-9. doi: 10.1073/pnas.1008150108. Epub 2011 Mar 30.
Within the field of eukaryotic protein synthesis, one factor remained putative for decades: eukaryotic translation initiation factor (eIF) 5A. Because eIF5A is an essential protein required for cell proliferation, and one easily targeted by inhibitors, identifying its role in the cell remains important and urgent. Recent reports support early findings that eIF5A stimulates protein synthesis and newly assign the factor a role in elongation rather than initiation. Here we show that eIF5A directly stimulates protein synthesis on native mRNAs, that rapid depletion of eIF5A in vivo immediately leads to a 2-fold inhibition of protein synthesis, and that both the immediate and lasting effects of eIF5A depletion are a reduction in polysome size concomitant with eIF5A depletion. Addition of purified eIF5A to a depleted lysate results in a roughly 2-fold stimulation of protein synthesis in vitro, a result consistent with both older methionyl-puromycin synthesis data and more recently published findings. We find that although eIF5A is not required for protein synthesis, it stimulates the process by about 2- to 3-fold. Our data, along with other published results, reinforce the conclusion that eIF5A stimulates protein synthesis with one important difference: Polysome profiles observed immediately after eIF5A depletion are diagnostic for a role in initiation. This discrepancy is discussed.
在真核生物蛋白质合成领域，有一个因素几十年来一直被认为是假设性的：真核翻译起始因子（eIF）5A。由于 eIF5A 是细胞增殖所必需的一种必不可少的蛋白质，并且很容易被抑制剂靶向，因此确定其在细胞中的作用仍然很重要和紧迫。最近的报告支持早期的发现，即 eIF5A 刺激蛋白质合成，并新赋予该因子在延伸而不是起始过程中的作用。在这里，我们表明 eIF5A 可直接刺激天然 mRNA 上的蛋白质合成，体内快速耗尽 eIF5A 会立即导致蛋白质合成抑制 2 倍，并且 eIF5A 耗尽的即时和持久影响都是与 eIF5A 耗尽伴随的聚核糖体大小减小。将纯化的 eIF5A 添加到耗尽的裂解物中会导致体外蛋白质合成大约 2 倍的刺激，这一结果与较早的甲硫氨酸嘌呤霉素合成数据和最近发表的发现一致。我们发现，尽管 eIF5A 不是蛋白质合成所必需的，但它可将该过程刺激大约 2-3 倍。我们的数据以及其他已发表的结果加强了 eIF5A 通过一种重要差异刺激蛋白质合成的结论：在 eIF5A 耗尽后立即观察到的多核糖体图谱可用于鉴定起始作用。讨论了这种差异。
