UC San Diego

Fluorescence and bioluminescence complementation assays have been commonly used to examine protein subcellular localization or behavior. This work outlines parallel fluorescence and bioluminescence complementation systems used in the assessment of any protein-of-interest’s (POI) translocation to the ER lumen. The fluorescence system, ERTrap, consists of one ER membrane “trapping” protein with a red fluorescent marker (Trap2) with translocation benchmarked against two control constructs: a cytosolic mNG11-mTagBFP construct (CytoBait) or an ER lumen mNG11-mTagBFP (ERBait). The bioluminescence system, BLERA, consists of orthogonal bioluminescent constructs to ERTrap. AMPK, a serine/threonine kinase promoting catabolic processes like autophagy, reportedly phosphorylates proteins with localization to the ER lumen like angiotensin-converting enzyme 2 (ACE2) and programmed death-ligand 1 (PD-L1). AMPK phosphorylation sites on PD-L1 (S195) and ACE2 (S680) are on the proteins’ extracellular domains, which would be accessible to AMPK if it was within the secretory pathway or extracellular. With no available studies on AMPK translocation and ER lumen substrate phosphorylation, ERTrap was developed to clarify if AMPK’s catalytic (α) or regulatory (β) subunit could enter the ER.No significant translocation of AMPKα or β subunit was observed, marked by low colocalization with ERTrap. Considering that AMPKα and β subunit entry may be in low amounts to escape fluorescent detection, the BLERA system with enhanced sensitivity was designed to allow for further testing of luminal AMPK translocation. Although our assays showed no significant luminal entry of AMPK, both tools could be used to quantify translocation or abundance of other POIs in the secretory pathway.