A proteome‐wide assay for protein mistargeting in living cells

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 ( cyt APEX) or an ER lumen‐retained horseradish peroxidase ( ER HRP) 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