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

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.

No abstract

Nascent chains of secretory proteins are targeted to the endoplasmic reticulum (ER) to start their journey in the secretory pathway. Yet mistargeting may not be rare, especially during stress. Mistargeted nascent proteins then accumulate in the cytosol, which can be a potential threat to the proteostasis of the cell. A comprehensive understanding of how protein gets mistargeted, which kinds of proteins are more prone to mistargeting and which factors govern the regulation of mistargeting will be important. However, those questions are hard to answer due to the lack of methodologies: the gold standard relying on in vitro transcription/translation is quantitative but tedious, low through‐put and incompatible with living cells, while the naïve proxy of measuring ER maturation events is facile but not necessarily accurate. Here, we developed a protein partitioning assay by using a cytosol‐localized engineered ascorbate peroxidase (cytAPEX) or an ER lumen‐retained horseradish peroxidase (ERHRP) to proximity‐label cytosolic and ER luminal proteomes respectively. The compartmental selectivity of this assay was validated with exogenous and endogenous proteins. We showed protein localization can be independent of maturation events, like signal sequence cleavage. We measured how an ER stressor impacts the accumulation of ER proteins in the cytosol. We also characterized differential protein targeting under the condition of translocon inhibition. Using quantitative proteomics, we calculated the basal import efficiency of some ER‐directed proteins. Interestingly, these data have good agreement with the in vitro method. With this assay, we will be able to estimate the basal import efficiency or measure mistargeting at a proteome‐wide scale in living cells. This assay will also make it possible to investigate the mechanism behind mistargeting in response to ER stress. Support or Funding Information University of California, Riverside

Most eukaryotic secretory proteins are co-translationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER Hsp70 Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin (TTR), leading to accumulation and aggregation of immature TTR in the cytosol. ATPase inactive mutants of Hspa13 further inhibit translocation and maturation of secretory proteins. While Hspa13 overexpression inhibits cell growth and ER quality control, HSPA13 knockout destabilizes proteostasis and increases sensitivity to ER disruption. Thus, we propose that Hspa13 regulates import through the translocon to maintain both ER and cytosolic protein homeostasis.The raw mass spectrometry data associated with this manuscript has been deposited in the PRIDE archive and can be accessed at PXD033498.