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

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Eukaryotic protein trafficking is vital for cellular function.
• Cellular stress can lead to protein mistargeting, disrupting protein homeostasis.
• Existing methods for studying protein mistargeting are not easily applicable to living cells.

Purpose of the Study:

• To develop and validate a novel proximity labeling technique for characterizing protein import into the endoplasmic reticulum (ER).
• To investigate stress-dependent protein mistargeting processes, including ER pre-emptive quality control (ER pQC).
• To identify cellular signaling networks influencing ER protein import.

Main Methods:

• Utilized genetically encoded peroxidases (ERHRP/cytAPEX) for multiplexed protein labeling and mistargeting identification.
• Employed transthyretin (TTR) as a known ER pQC substrate.
• Investigated the impact of ERHRP expression levels on TTR aggregation and labeling efficiency.
• Assessed TTR mistargeting under conditions of Sec61 inhibition and ER stress.
• Examined the effect of ATF6 activation on TTR import.

Main Results:

• The ERHRP/cytAPEX pair demonstrated high selectivity and sensitivity for protein labeling and mistargeting detection.
• Low ERHRP expression minimized TTR aggregation without compromising labeling efficiency.
• CytAPEX labeling successfully identified TTR mistargeting caused by Sec61 inhibition and ER stress-induced ER pQC.
• Stress-free activation of ATF6 mimicked the TTR import deficiency observed in ER pQC.

Conclusions:

• Proximity labeling with genetically encoded peroxidases is an effective strategy for studying ER protein import in living cells.
• This method allows for the characterization of factors influencing protein trafficking under various cellular conditions.
• The study provides new insights into the mechanisms of stress-dependent protein mistargeting and ER quality control.