UC Riverside

Nearly all proteins are synthesized in the cytosol. A third of this proteome must be trafficked elsewhere, such as to membranes, to subcellular compartments, or outside of the cell. Proper trafficking of nascent protein is necessary for protein folding, maturation, quality control and cellular and organismal health. 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. Proximity labelling has been a powerful approach for characterizing subcellular proteomes. Herein, we use genetically encoded heme peroxidases to characterize protein import into the endoplasmic reticulum (ER). Firstly, we show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for multiplexed protein labelling, and for identifying protein mistargeting, using a known ER pQC substrate transthyretin (TTR). cytAPEX labelling recovers mistargeted TTR due to translocon blockade or ER pQC. Furthermore, we discover that stress-free activation of the ER unfolded protein response (UPR) sensor ATF6 mistargets TTR. We also evaluate ER stressors tunicamycin, Brefeldin A and DTT in HEK293T cells. Only thapsigargin induces substantial TTR mistargeting, while the result of other stressors remains ambiguous due to limited sensitivity. To enable a more quantitative platform for ER import, we integrate parallel reaction monitoring mass spectrometry. With this approach, we revisit the relationship between ER stressors and ER pQC. Indeed, only ER calcium depletion by thapsigargin or cyclopiazonic acid mistargets the most TTR, suggesting UPR itself is not sufficient. We also evaluate different normalization factors for relative quantification - MS1 total ion current, cytAPEX, pyruvate carboxylase, actin, etc. We consider that labelling activity (e.g. cytAPEX) as the most appropriate standard, because labelling activity may be subjected to change due to drug treatment. The last session includes efforts to expand this assay for proteome-wide protein import measurement. Our initial attempts may be obscured due to prolonged drug treatments, their pleiotropic effects, and a cell type that does not secrete much protein. To overcome those limitations, we generate HepG2 stable cell lines with either heme peroxidase. Drug treatment is shortened to 2 hours, and translation maintained by integrated stress response inhibitor ISRIB.