PDF(Pubmed)
Abstract：
Proximity labeling with genetically encoded enzymes is widely used to study protein-protein interactions in cells. However, the accuracy of proximity labeling is limited by a lack of control over the enzymatic labeling process. Here, we present a light-activated proximity labeling technology for mapping protein-protein interactions at the cell membrane with high accuracy and precision. Our technology, called light-activated BioID (LAB), fuses the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. We demonstrate, in multiple cell lines, that upon illumination with blue light, CRY2 and CIB1 dimerize, reconstitute split-TurboID and initiate biotinylation. Turning off the light leads to the dissociation of CRY2 and CIB1 and halts biotinylation. We benchmark LAB against the widely used TurboID proximity labeling method by measuring the proteome of E-cadherin, an essential cell-cell adhesion protein. We show that LAB can map E-cadherin-binding partners with higher accuracy and significantly fewer false positives than TurboID.
摘要：
遗传编码酶的邻近标记被广泛用于研究细胞中的蛋白质-蛋白质相互作用。然而,邻近标记的准确性受到缺乏对酶标记过程的控制的限制。这里,我们提出了一种光活化邻近标记技术，用于高精度和高精度地绘制细胞膜上蛋白质-蛋白质相互作用的图谱。我们的技术,称为光激活生物ID（LAB），将分裂的TurboID邻近标记酶的两半融合到光二聚体蛋白CRY2和CIB1上。我们证明,在多个细胞系中，在蓝光照射下，CRY2和CIB1二聚化，重组split-TurboID并启动生物素化。关灯导致CRY2和ClB1的解离并停止生物素化。我们通过测量E-cadherin的蛋白质组，将LAB与广泛使用的TurboID邻近标记方法进行基准测试，一种必需的细胞-细胞粘附蛋白。我们表明，LAB可以比TurboID更高的准确性和更少的假阳性来映射E-cadherin结合伴侣。
