The effects of E2 enzyme mutations on the glutamate receptor GLR-1 in the endoplasmic reticulum-associated protein degradation …

When proteins are misfolded, they may lose their ability to function correctly, accumulate within a cell, and form protein aggregates that disrupt other processes. Therefore, the ability to degrade misfolded proteins is important for maintaining healthy cellular function. Endoplasmic reticulum-associated degradation (ERAD) is a ubiquitin-dependent process that allows cells to eliminate misfolded proteins and prevent toxic protein aggregation at the Endoplasmic Reticulum (ER). UBC-6 and UBC-7 are E2 ubiquitin-conjugating enzymes that are important for ERAD in yeast, but there is much less known about they may work during ERAD in neurons. We use mutational analyses to understand how vital UBC-6 and UBC-7 are in regulating neurotransmitter receptors in the nematode worm, C. elegans. We used two types of mutations: point mutations and gene knockouts. We hypothesized that a gene knockout of ubc-6 or ubc-7 could cause an increase in protein aggregation when compared with single point mutations because a larger deletion may be more severe and has been associated with behavioral defects, while a point mutation might leave the enzyme still active. In order to track neural protein aggregation, we follow the glutamate receptor GLR-1 with a green fluorescent protein (GFP) tag (GLR-1::GFP) and utilize fluorescence microscopy and FIJI image analyzing software. We found that ubc-7 knockout mutants had a significantly higher abundance of GLR-1::GFP in cells that express the fusion protein when compared to wild-type and the other mutations. Our findings suggest that our point mutation mutants and ubc-6 mutants still carry out ERAD, though less efficiently than wild-type. Since the knockout mutations in UBC-7 resulted in increased protein aggregation when compared with UBC-6, this suggests that UBC-7 is more vital for ERAD than UBC-6 in regulating neurotransmitter receptors.