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

Abstract

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), are 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 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 demonstrate 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.

Hspa13 Regulates Endoplasmic Reticulum and Cytosolic Proteostasis Through Modulation of Protein Translocation

“…Also, this assay copurifies cytosolic aggregates to the microsomal population (49). To better support cytosolic assignment for immature Flag TTR, we applied our recently reported assay, which uses proximity labeling to measure ER protein mistargeting (46). Specifically, horseradish peroxidase with an N-terminal preimmunoglobulin signal sequence and C-terminal KDEL ER retention sequence ( ER HRP) localizes to the ER (50), while an optimized ascorbate peroxidase ( cyt APEX2) localizes to the cytosol (51).…”

“…Another possibility is that Hspa13 inhibits signal peptide cleavage directly, with immature Flag TTR then being rapidly retrotranslocated to the cytosol through ER-associated degradation (ERAD). However, we have previously demonstrated the inhibition of TTR signal peptide cleavage in the ER does not induce its cytosolic accumulation (46), but rather leads to ER accumulation of immature TTR instead.…”

“…Proximity Labeling. We used ER HRP and cyt APEX2 labeling to biotinylate proteins that are ER-lumenal space and cytosol targeted, respectively (46,117,118). Unless otherwise indicated, cells were treated with 1 µM MG132 treatment for 16 h prior to harvesting to inhibit proteasomal degradation.…”

“…Transthyretin (TTR) is a homotetrameric secretory protein that is frequently used as a model substrate for ER protein homeostasis studies due to its extraordinarily well-characterized biophysical parameters, high secretory load, and disease relevance (40)(41)(42). TTR is particularly well-suited to ER import experiments, as a small population basally mistargets to the cytosol, this population increased with ER stress, and the immature and mature forms are readily separable by SDS-PAGE gel (43)(44)(45)(46). To determine whether Hspa13 affects TTR localization, we co-overexpressed HEK293T cells with Hspa13 and Flag-tagged TTR ( Flag TTR), with the Flag tag immediately C-terminal to the TTR signal peptide.…”

“…We also compared the effect of Hspa13 overexpression to UPR induction by thapsigargin (Tg), which promotes secretion of aggregation-prone conformations of TTR (79). While Tg upregulates the UPR target BiP, and modestly inhibits TTR import efficiency (44,46), it does not increase insoluble TTR to a similar extent as Hspa13 overexpression does (Figure S4D).…”