Methodologies for Measuring Protein Trafficking across Cellular Membranes

Lyu, Ziqi; Genereux, Joseph C.

doi:10.1002/cplu.202100304

Cited by 4 publications

(5 citation statements)

References 191 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This process is termed ER pre-emptive quality control 11,12 (sometimes denoted ER pQC; we use ER pre-QC instead, to avoid confusion with generic protein quality control PQC [13][14][15] ). Because current techniques for measuring protein mislocalization are onerous, semi-quantitative, or in vitro 16,17 , we do not yet have clear understanding of the substrates and biochemical mechanisms of ER pre-QC 18 , nor which stresses activate it.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

Lyu

Genereux

2023

Preprint

Self Cite

View full text Add to dashboard Cite

Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which is only semi-quantitative and suffers from poor sensitivity. Here, we integrate parallel reaction monitoring mass spectrometry to enable a more quantitative platform for ER import. PRM as opposed to densitometry improves quantification of transthyretin mistargeting while also achieving at least a ten-fold gain in sensitivity. The multiplexing of PRM also enabled us to evaluate a series of normalization approaches, revealing that normalization to auto-labeled APEX2 peroxidase is necessary to account for drug treatment-dependent changes in labeling efficiency. We apply this approach to systematically characterize the relationship between chemical ER stressors and ER pre-QC induction in HEK293T cells. Using dual-FLAG-tagged transthyretin (FLAGTTR) as a model secretory protein, we find that Brefeldin A treatment as well as ER calcium depletion cause pre-QC, while tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform.

show abstract

Section: Introductionmentioning

confidence: 99%

Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

Lyu

Genereux

2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…This process is termed ER pre-emptive quality control (Kang et al, 2006;Kadowaki et al, 2015) (sometimes denoted ER pQC; we use ER pre-QC instead, to avoid confusion with generic protein quality control PQC (McCaffrey and Braakman, 2016;Arrieta et al, 2017;Schwabl and Teis, 2022)). Because current techniques for measuring protein mislocalization are onerous or in vitro (Lyu and Genereux, 2021;Sharma et al, 2010), we do not yet have clear understanding of the substrates and biochemical mechanisms of ER pre-QC (Kadowaki and Nishitoh, 2019), nor which stresses activate it.…”

Section: Introductionmentioning

confidence: 99%

Quantitative measurement of transthyretin mistargeting by proximity labeling and parallel reaction monitoring

Lyu,

Genereux

2023

Front. Chem. Biol.

Self Cite

View full text Add to dashboard Cite

Introduction: Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which sometimes suffers from poor sensitivity.Methods: Here, we integrate parallel reaction monitoring (PRM) mass spectrometry to enable a more quantitative platform and assess how chemical ER stressors impact pre-QC of the model secretory protein transthyretin in HEK293T cells.Results and Discussion: We find that some drug treatments affect labeling efficiency, which can be controlled for by normalizing to APEX2 autolabeling. While some chemical ER stress inducers including Brefeldin A and thapsigargin induce pre-QC, tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform.

show abstract

“…ER stress upregulates ERAD, lowering the ER misfolded protein burden. ER stress can also protect the ER by decreasing nascent protein import, through the pre-emptive quality control (ER pQC) pathway. − Mistargeting of secretory proteins to the cytosol can be deleterious for that compartment, however, and has been implicated in neurodegeneration and diabetes. − A lack of high-throughput methods for measuring protein targeting efficiency in cells has challenged full characterization of secretory protein mistargeting …”

Section: Introductionmentioning

confidence: 99%

“…15−18 A lack of high-throughput methods for measuring protein targeting efficiency in cells has challenged full characterization of secretory protein mistargeting. 19 The gold standard technique for quantifying protein import into the ER is the microsomal import assay based on proteinase K protection. 5,20 In this method, nascent protein is metabolically 35 S-labeled, usually as part of an in vitro transcription/translation formulation in the presence of freshly prepared pancreatic microsomes.…”

Section: ■ Introductionmentioning

confidence: 99%

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

et al. 2022

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Methodologies for Measuring Protein Trafficking across Cellular Membranes

Cited by 4 publications

References 191 publications

Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

Quantitative measurement of transthyretin mistargeting by proximity labeling and parallel reaction monitoring

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

Contact Info

Product

Resources

About