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  • A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast.

A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast.

Cell chemical biology (2020-05-30)
Yi Li, Caiping Tian, Keke Liu, Ying Zhou, Jing Yang, Peng Zou
ABSTRACT

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.

MATERIALS
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Product Description

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