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Published on: August 2, 2015
A Clickable APEX Probe for Proximity-Dependent Proteomic Profiling in Yeast.
Yi Li1, Caiping Tian2, Keke Liu2
1College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Peking University, Beijing 100871, China.
Researchers developed Alk-Ph, a novel substrate that significantly enhances engineered ascorbate peroxidase (APEX) labeling efficiency in yeast. This breakthrough improves proximity-dependent protein and RNA mapping in microbial systems.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Engineered ascorbate peroxidase (APEX) enables proximity-dependent labeling of proteins and RNAs in live cells.
- APEX applications in microorganisms are limited by inefficient biotin-phenol (BP) substrate labeling.
- Improving substrate permeability and labeling efficiency is crucial for microbial APEX studies.
Purpose of the Study:
- To design and screen novel alkyne-functionalized substrates for enhanced APEX labeling in yeast.
- To improve cell wall permeability and labeling efficiency compared to the standard BP substrate.
- To expand the utility of APEX for spatial proteome and transcriptome mapping in yeast.
Main Methods:
- Screening of alkyne-functionalized substrates for APEX-mediated labeling in yeast.
- Utilizing protein-centric and peptide-centric chemoproteomic experiments.
- Assessing proximity-dependent RNA labeling using the APEX system.
Main Results:
- A novel substrate, Alk-Ph, demonstrated substantially improved APEX labeling efficiency in intact yeast cells.
- Alk-Ph exhibits enhanced cell wall permeability compared to the traditional biotin-phenol (BP) substrate.
- Identification of 165 yeast mitochondrial matrix proteins with 94% specificity.
- Demonstration of Alk-Ph's utility for proximity-dependent RNA labeling, expanding APEX-seq applications.
Conclusions:
- Alk-Ph represents a significant advancement for APEX-based proximity labeling in yeast.
- The improved labeling strategy facilitates large-scale spatial proteome and transcriptome mapping in yeast.
- This work overcomes previous limitations, enabling broader APEX applications in microbial research.
