Dynamic Mapping of Proteome Trafficking Within and Between Living Cells by TransitID

Authors

Wei Qin
Joleen S Cheah
Charles Xu
James Messing
Brian D Freibaum
Steven Boeynaems
J Paul Taylor
Namrata D Udeshi
Steven A Carr
Alice Y Ting

Publication Date

7-20-2023

Journal

Cell

DOI

10.1016/j.cell.2023.05.044

PMID

37385249

PMCID

PMC10527209

PubMedCentral® Posted Date

7-20-2024

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

Keywords

Proteome, Mitochondria, Cell Nucleolus, Mass Spectrometry, Gene Expression Regulation

Abstract

The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.