Description

Maintaining proteostasis, or protein homeostasis, is an important cellular function because misfolded proteins can aggregate and contribute to neurodegenerative diseases. One way that cells preserve proteostasis is through the Endoplasmic Reticulum Associated Degradation pathway (ERAD). ERAD relies on interactions between E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases to ubiquitylate misfolded proteins in order to signal for their destruction via the proteasome (Vembar and Brodsky 2008). We investigated if missense mutations in genes that encode two E2 ubiquitin-conjugating enzymes in C. elegans, UBC-6 and UBC-7, affect spontaneous reversal frequency (Brockie et al. 2001; Jones et al. 2002; Stewart et al. 2016; Zheng et al. 1999).

UBC-6 and UBC-7 are conserved across Eukaryotes (Figure 1C). We obtained strains of animals from the Million Mutation Project with the missense mutations that confer the amino acid changes T158I in UBC-6 and P79L in UBC-7. The analogous residues in human Ube2j1 or Ube2j2 and Ube2g2 are Asp160 or 153 and Proline 78, respectively, which were mapped onto the available structures of the human enzymes (Figure 1C). In Ube2j2 (C. elegans UBC-6) Asp153 makes contact with Phe156, and mutating this residue decreases the efficiency of the enzyme (Tobi Ritterhoff, personal communication). Structural alignments suggest that Pro78 (mutated in UBC-7) is part of the well-conserved HPN (Histadine-Proline-Asparagine) motif, so mutation of the analogous residue was hypothesized to abrogate enzymatic activity (Cook and Shaw 2012; Wu et al. 2003).

Our results show that the E2 ubiquitin-conjugating enzyme mutations did not significantly affect C. elegans spontaneous reversal frequency (Figure 1A). glr-1 animals were used as a control and reversed significantly less frequently than wild-type animals, as previously reported (Kowalski et al. 2011). Both ubc-6 and ubc-7 animals had less frequent spontaneous reversals than WT animals and more frequent spontaneous reversals than glr-1 animals, but neither of these results were significant.

One clear reason for the insignificant change in reversal frequency in the ubc-6 and ubc-7 mutant animals could be that the missense mutations in these strains we observed do not affect gene function. It is also possible that UBC-6 and UBC-7 are able to compensate for each other in C. elegans. In S. cerevisiae Ubc6p and Ubc7p work cooperatively and sequentially to target substrates in coordination with the E3 ligases Doa10p (orthologous to C. elegans MARC-6), and Hrd1p (orthologous to C. elegans HRD-1 and paralogous to HRDL-1) (Sasagawa et al. 2007; Weber et al. 2016). It could be that this mechanism of tandem ubiquitylation is not conserved in C. elegans despite its conservation for human UBE2J2 (Weber et al. 2016). The human orthologs HRD1 and UBE2J1 (an additional ortholog of Ubc6p) also work together to target substrate proteins for degradation (Bays et al. 2001; Burr et al. 2011). Therefore it is possible that C. elegans E3s could collaborate with either UBC-6 or UBC-7 to target substrate proteins for degradation. In the future, we plan to address these possibilities using double ubc-6; ubc-7 point mutants and CRISPR-Cas9-targeted deletion mutants for UBC-6 and UBC-7, in combination with E3 ligase mutants.