The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely use in mammalian cells, APEX applications in microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX-labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP. Through a combination of protein-centric and peptide-centric chemoproteomic experiments, we have identified 165 proteins with a specificity of 94% in the yeast mitochondrial matrix. In addition, we have demonstrated that Alk-Ph is useful for proximity-dependent RNA labeling in yeast, thus expanding the scope of APEX-seq. We envision that this improved APEX-labeling strategy would set the stage for the large-scale mapping of spatial proteome and transcriptome in yeast.

工程化的抗坏血酸过氧化物酶（APEX）是一种功能强大的工具，可对活细胞中的蛋白质和RNA进行邻近依赖性标记。尽管APEX在哺乳动物细胞中得到了广泛使用，但由于其生物素-酚（BP）底物的标记效率低，在微生物中的APEX应用受到了阻碍。在这项研究中，我们试图通过设计和筛选一组炔官能化的底物来应对这一挑战。我们最好的探针Alk-Ph可以显着提高完整酵母细胞中APEX的标记效率，因为它比BP具有更高的细胞壁渗透性。通过以蛋白质为中心和以肽为中心的化学旋转实验的结合，我们在酵母线粒体基质中鉴定了165种具有94％特异性的蛋白质。此外，我们已经证明Alk-Ph可用于酵母中邻近依赖性RNA标记，从而扩大了APEX-seq的范围。我们设想，这种改进的APEX标记策略将为酵母中空间蛋白质组和转录组的大规模作图奠定基础。