The Effect of CRISPR Deletions of ubc-6 E2 Enzyme and sel-11 Enzyme on GLR-1 Localization

The Endoplasmic Reticulum (ER) uses a cellular system known as Endoplasmic Reticulum Associated-Degradation (ERAD) to detect and degrade misfolded proteins. The ERAD associated enzymes ubc-6 (E2) and sel-11 (E3) enzymes ubiquitylate the misfolded proteins and tag them for degradation. The Unfolded Protein Response (UPR) is activated when misfolded proteins accumulate and is known to participate in the proper cellular trafficking of the transmembrane protein GLR-1, a glutamate receptor. We are studying the effects of gene deletions using the CRISPR/Cas9 system of ubc-6 and sel-11, on the accumulation of GLR-1 in Caenorhabditis elegans. We hypothesize that because E2 enzymes work upstream and in the same pathway as E3 enzymes, ubc-6 mutants would have higher levels of GLR-1 accumulation when compared to the sel-11 mutants. PCR genotyping was used to confirm the CRISPR mediated deletion was successful and the genotype of the nuIs24 strain (wildtype, WT) was confirmed. The mutant sel-11 and ubc-6 strains were crossed with the genotyped WT to compare the GLR-1 protein levels to the WT. Western Blotting was used to confirm genotypes and quantify GLR-1::GFP levels in each of the strains. Fluorescence microscopy was used to visualize the location of GLR-1::GFP in the mutants crossed with the nuIs24 and compare to WT. Our experiments found GLR-1::GFP levels were higher in the WT strain than in either of the mutant crosses. Western Blots also showed that GLR-1::GFP levels in the E3 mutant were similar to the E2 mutant. This data suggests that there are other proteins assisting in the degradation of the GLR-1 protein and further experiments are needed.