Negrutskii B S, Deutscher M P
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4991-5. doi: 10.1073/pnas.88.11.4991.
Channeling, the direct transfer of metabolic intermediates from one enzyme to another in a pathway, has received increased attention as an explanation for the high efficiency of cellular processes. The known structural organization of the protein biosynthetic machinery, and a recent suggestion that aminoacyl-tRNAs may be channeled, has led us to devise a direct test of this possibility. By employing the technique of electroporation, conditions were established for the introduction of aminoacyl-tRNAs into Chinese hamster ovary (CHO) cells. We show, by coelectroporation of various combinations of free [14C]amino acids and [3H]aminoacyl-tRNAs, that whereas the free amino acids serve as effective precursors for protein synthesis, the exogenous aminoacyl-tRNAs are utilized poorly, if at all. The lack of incorporation into protein from added aminoacyl-tRNAs is not due to their leakage from the cell, to their instability, or to their damage during electroporation. Furthermore, in contrast to the findings with intact cells, extracts of CHO cells incorporate both free amino acids and aminoacyl-tRNAs into protein with similar efficiencies. Based on these observations, we conclude that the inability of exogenous aminoacyl-tRNAs to serve as precursors for protein synthesis is due to the structural organization of intact cells that leads to channeling of this substrate in vivo. Thus, we propose that endogenously synthesized aminoacyl-tRNA is directly transferred from aminoacyl-tRNA synthetase to elongation factor to ribosome without dissociation into the cell fluid, and as a consequence, usage of exogenously introduced molecules is precluded.
通道化作用，即代谢中间产物在一条途径中从一种酶直接转移至另一种酶，作为细胞过程高效性的一种解释已受到越来越多的关注。蛋白质生物合成机制的已知结构组织，以及最近关于氨酰 - tRNA可能存在通道化作用的推测，促使我们对这种可能性进行直接测试。通过采用电穿孔技术，建立了将氨酰 - tRNA导入中国仓鼠卵巢（CHO）细胞的条件。我们通过对游离[¹⁴C]氨基酸和[³H]氨酰 - tRNA的各种组合进行共电穿孔实验表明，虽然游离氨基酸可作为蛋白质合成的有效前体，但外源性氨酰 - tRNA即使被利用，效率也很低。添加的氨酰 - tRNA未能掺入蛋白质并非由于其从细胞中泄漏、不稳定或在电穿孔过程中受损。此外，与完整细胞的实验结果相反，CHO细胞提取物能以相似的效率将游离氨基酸和氨酰 - tRNA掺入蛋白质。基于这些观察结果，我们得出结论，外源性氨酰 - tRNA不能作为蛋白质合成前体的原因是完整细胞的结构组织导致该底物在体内发生通道化作用。因此，我们提出内源性合成的氨酰 - tRNA直接从氨酰 - tRNA合成酶转移至延伸因子再到核糖体，而不会解离到细胞液中，结果是外源性引入的分子无法被利用。
