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 ERHRP/cytAPEX 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 ERHRP labeling induces formation of detergent-resistant
TTR aggregates, this is minimized by using low ERHRP expression,
without loss of labeling efficiency. cytAPEX 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.