Mahajan N P, Linder K, Berry G, Gordon G W, Heim R, Herman B
Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill 27599, USA.
Nat Biotechnol. 1998 Jun;16(6):547-52. doi: 10.1038/nbt0698-547.
It has been hypothesized that interaction of Bcl-2 and Bax may regulate apoptosis. The spatial and temporal interaction of Bcl-2 and Bax at the single cell level has not, however, been demonstrated. To achieve this goal, we have developed two-fusion FRET (fluorescence resonance energy transfer). Using green fluorescent protein (GFP)-Bax and blue fluorescent protein (BFP)-Bcl-2 fusion proteins coexpressed in the same cell, we demonstrate a direct interaction between Bcl-2 and Bax in individual mitochondria. Mitochondrially localized cytochrome c-GFP and BFP-Bcl-2 showed little or no FRET, while nuclear-localized GFP-human papillomavirus E6 and BFP-Bcl-2 did not interact when coexpressed in the same cell. These findings indicate that two-fusion FRET provides an opportunity to examine the interaction between two different proteins coexpressed in single intact mammalian cells.
据推测，Bcl-2与Bax的相互作用可能调节细胞凋亡。然而，Bcl-2与Bax在单细胞水平上的时空相互作用尚未得到证实。为实现这一目标，我们开发了双融合荧光共振能量转移技术（FRET）。通过在同一细胞中共表达绿色荧光蛋白（GFP）-Bax和蓝色荧光蛋白（BFP）-Bcl-2融合蛋白，我们证明了在单个线粒体中Bcl-2与Bax之间存在直接相互作用。线粒体定位的细胞色素c-GFP和BFP-Bcl-2几乎没有显示出FRET现象，而核定位的GFP-人乳头瘤病毒E6和BFP-Bcl-2在同一细胞中共表达时不发生相互作用。这些发现表明，双融合FRET为研究在单个完整哺乳动物细胞中共表达的两种不同蛋白质之间的相互作用提供了一个契机。
