Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling.

The proper trafficking of eukaryotic proteins is essential to cellular function. Genetic, environmental, and other stresses can induce protein mistargeting and, in turn, threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), is difficult to parse. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the HRP/APEX pair provides good selectivity and sensitivity for both multiplexed protein labeling and for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although HRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low HRP expression, without loss of labeling efficiency. APEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we discover that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.