ER-phagy (reticulophagy) is a selective autophagic process that clears damaged endoplasmic reticulum (ER), ER membranes, or proteins to maintain cellular homeostasis when the function of ER is altered. ER plays crucial roles in various cellular processes such as protein folding, modification, lipid and hormone synthesis, ion storage, and metabolic regulation. However, unfolded or misfolded proteins accumulation within the ER disrupts its homeostasis under internal or external stimuli. To accelerate the degradation of ER-associated proteins (ERAD), cells initiate a self-protective program called the unfolded protein response (UPR). Prolonged ER stress and UPR can trigger ER-phagy to mantain ER homeostasis. Various human diseases including neurodegenerative diseases, cancer, and metabolic disorders are associated with ER-phagy. Therefore efficient tools for monitoring ER-phagy activity are significant for elucidating molecular mechanisms and physiological/pathological effects as well as identifying drugs.
Tools | Function | Product Offering |
mCherry-EGFP-RAMP4 | ER-phagy | LV/Ad/AAV |
mCherry-RAMP4 | ||
ssmRFP1-EGFP-KDEL |
*Note: LV: Lentivirus, Ad: Adenovirus, AAV: Adeno-associated virus.
In the cytoplasm, co-expression of red and green fluorescent proteins leads to the generation of yellow punctas. Upon induction of ER-phagy, the acidic environment within lysosomes quenches the green fluorescence, leaving only the red fluorescence intact. The quantification of red fluorescent punctas enables monitoring of ER-phagy.
PMID:30143524 IF: 7.8 Q1 (mCherry-EGFP-RAMP4 tool)
PMID: 31006538 IF: 16.0 Q1 (ssmRFP1-EGFP-KDEL tool)
Moreover, in the lysosomes, cleavage occurs between mRFP1 and EGFP in the ssmRFP1-EGFP-KDEL tool. The determination of mRFP1/mRFP1-EGFP ratio through Western Blot analysis provides an indication for ER-phagy.
PMID: 31006538 IF: 16.0 Q1 (ssmRFP1-EGFP-KDEL tool)
In the cytoplasm, expression of fusion protein mCherry-RAMP4 takes place; upon initiation of ER-phagy, hydrolytic enzymes cleave this fusion protein to release the mCherry fluorescence protein. Calculation of mCherry/mCherry-RAMP4 band intensity ratio from Western Blot results serves as an indicator for ER-phagy.
PMID: 30143524 IF: 7.8 Q1 (mCherry-RAMP4 tool)
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