Description

Secretory and membrane-bound proteins must be properly folded and matured at the endoplasmic reticulum (ER). Despite the molecular machinery dedicated to these processes, up to 1/3 of proteins are destroyed within minutes of their synthesis (Hirsch et al. 2009; Schubert et al. 2000). Misfolded proteins in the endoplasmic reticulum can accumulate and disrupt proteostasis, which can contribute to neurodegenerative diseases. The Endoplasmic Reticulum Associated Degradation (ERAD) pathway relies on E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases to ubiquitylate misfolded proteins, signaling for degradation of these misfolded proteins by the proteasome (Vembar and Brodsky 2008). Three putative E3 ligases that are expected to be involved in ERAD in C. elegans are HRDL-1, HRD-1, and MARC-6 (Sasagawa et al. 2007). We used strains harboring mutations in hrdl-1, hrd-1 and marc-6 genes to determine if these proteins are required for regulating spontaneous reversal behavior in C. elegans.

Spontaneous reversals are a C. elegans behavior whose frequency is regulated by well-defined circuitry and neurotransmitter receptors, including the glutamate receptor, glr-1 (Brockie et al. 2001; Burbea et al. 2002; Dahlberg and Juo 2014, Hart et al. 1995; Kowalski et al. 2011; Zheng et al. 1999). We hypothesized that the spontaneous reversal frequency behavior of C. elegans would be affected by E3 ligase mutations if they are important for normal spontaneous reversal behavior. glr-1 animals reverse significantly less than wild-type animals and were used as a positive control (Figure 1B) (Hart et al. 1995; Kowalski et al. 2011). Animals harboring mutations in marc-6 and hrd-1 also reverse significantly less than wild-type animals (Figure 1A). Animals lacking full-length hrdl-1 reversed less than wild-type animals, but this was not statistically significant (Figure 1A).

The primary motivation for our work was to ask if the glutamate receptor, GLR-1, might be regulated by these E3s, but further research must be done in order to determine molecular mechanisms that cause the differences in behavior that we report. Because ERAD E2 and E3 proteins can compensate for each other’s absence, we hypothesize that in the absence of any one E3 ligase, others are upregulated either through protein activity or gene expression (Bays et al. 2001; Weber et al. 2016). For example, if HRD-1 is upregulated to compensate for the putative loss-of-function of HRDL-1 in hrdl-1 mutant animals, this could explain why hrdl-1 animals did not show a statistically significant reduction in reversals/minute compared to wild-type animals. However, our results from the hrd-1 and marc-6 mutants suggest that there is not complete redundancy between the E3 ligases. Future experiments will focus on testing this hypothesis using double and triple mutants in the E3 ligase genes.