Interfaculty Institute of Biochemistry, University of Tübingen, Germany.
Proteome Center Tübingen, University of Tübingen, Germany.
FEBS J. 2020 Jan;287(2):325-344. doi: 10.1111/febs.15007. Epub 2019 Aug 12.
Enzyme-catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin-phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane-enclosed and a semiopen compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. Its human ortholog, melanocyte proliferating gene 1, is linked to developmental processes and dermatological diseases. A first characterization of the Yer156C neighborhood reveals an array of proteins linked to proteostasis and RNA binding. Thus, our approach establishes APEX2 PL as another powerful tool that complements the methods palette for the model system yeast.
酶催化的邻近标记（PL）与工程化的抗坏血酸过氧化物酶 APEX2 是一种在活细胞中绘制细胞器区室化和蛋白质网络的新方法。目前为哺乳动物细胞开发的程序不允许将辅助底物生物素-苯酚递送到活酵母细胞中。在这里，我们提出了一种基于半透酵母细胞的新方法。与稳定同位素标记的氨基酸在细胞培养（SILAC）相结合，我们展示了膜封闭和半开放隔室、线粒体基质和核的蛋白质组图谱。APEX2 PL 揭示了以前通过常规技术未鉴定出的核蛋白。其中之一，Yer156C 蛋白高度保守但功能未知。其人类同源物黑素细胞增殖基因 1 与发育过程和皮肤疾病有关。对 Yer156C 邻近区域的首次表征揭示了一系列与蛋白质稳态和 RNA 结合相关的蛋白质。因此，我们的方法确立了 APEX2 PL 作为另一种强大的工具，补充了酵母模型系统的方法组合。
