microPublication Biology2578-9430Caltech Library10.17912/micropub.biology.000328New FindingPhenotype DataC. elegans
Individual point mutations in two ERAD E2 ubiquitin-conjugating enzymes do not affect
Caenorhabditis elegans
spontaneous reversal frequency
Oswald Mackenzi 1Hulsey-VincentHeino1DahlbergCaroline (Lina)1§
Department of Biology, Western Washington University, Bellingham, WA, 98225, USA
LuthEric
Correspondence to: Caroline (Lina) Dahlberg (
lina.dahlberg@wwu.edu
)
MO: Formal analysis, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing, Methodology
HH: Formal analysis, Visualization, Writing - review and editing
A.
Spontaneous reversal frequency assays were performed using wild type (WT)
C. elegans
, alongside strains harboring mutations in
glr-1
or in genes that encode the E2 ubiquitin-conjugating enzymes,
ubc-6
and
ubc-7
. Box and whisker plots show the average reversals per minute, bounded by quartiles; the line in each box represents the median of the average reversals per minute for each genotype. N=20 individual animals for all genotypes; significance relative to WT was calculated using the Tukey-Kramer test following a one-way ANOVA (*, p=0.0027 for
glr-1
; non-significant: p=0.58 for
ubc-6
; p=0.48 for
ubc-7
). The data is normally distributed (Shapiro-Wilk test, p=0.86) and groups show equal variance (residuals vs. fitted plot).
B.
Schematics of E2 ubiquitin-conjugating enzyme genes and
glr-1
gene, including the locations of point mutations. Schematics were made using http://wormweb.org/exonintron.
C.
Crystal structures of the human E2 Ube2j2 (top, with rotated view to show the back side of the protein) and Ube2g2 (bottom, left). Models were made using Pymol. Residues that are mutated are shown in red (Ube2j2, D153; UBC-6, T158) and yellow (Ube2g2, P78, UBC-7, P79). Residue F156 of Ube2j2 is shown in cyan. Alignments of
C. elegans
UBC-6 and UBC-7 regions of homology, compared to putative orthologs in other eukaryotes. The highlighted residues correspond to those shown in the structures.
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.
Methods
Reversal assay protocol
: Each
C. elegans
strain was grown on separate NGM agar plates seeded with OP50, at 21.4°C. While setting up for the reversal assays, multiple young adult
hermaphroditic nematodes from each strain were picked onto separate seeded NGM agar plates and coded. This allows for blind analysis of the videotaped trials to occur when scoring the reversal assays, in order to reduce potential bias. During the assay, one animal at a time was picked using halocarbon oil (a non-food substance) from its coded seeded plate onto an unseeded NGM agar plate to induce food-seeking behavior. The animal was allowed to move around for two minutes before videotaping began. If the animal did not move away from its initial position after two minutes it was discarded and not counted in the data set. Each animal was recorded for five minutes and then discarded. At least one individual of each strain was observed during each experimental session in order to allow for any potential variations in temperature and humidity to be accounted for equally across all strains. The total N values listed represent measurements from multiple experimental sessions.
Scoring reversals
: After finishing recording all reversal assay trials for an experiment, the coded trials were viewed and scored blindly by one researcher. Movement was counted as a reversal if the tip of the nose moved backwards for at least 1/6th of the animal’s body length. The position of the posterior pharyngeal bulb was used as a marker for that distance.
Recording setup
: An Olympus SZ61 microscope attached to a TLB 4000 Series Substage Illuminator base was used to record the videos. The microscope was connected to The Imaging Source DFK 31AF03 color camera, which was connected to a computer running Windows OS. The software used to record the videos was Debut Professional by NCH Software.
Genotyping
: The genotypes of all strains were confirmed after completing the trials. The genotypes of KP4, CLD41 and CLD43 were confirmed using PCR and DNA sequencing.
Additional point mutations from the MMP were considered for these experiments (http://genome.sfu.ca/gexplore/) but only one other available allele (
ubc-6
(gk549681)) was also predicted to have an effect on enzyme function (Tobi Ritterhoff, personal communication). Although that strain was viable, we were unable to backcross it to remove background mutations. Other existing deletion mutants remove sequences outside of the coding locus of each gene. We also note that following our experiments, CRISPR-Cas9-targeted deletion mutants became available for each gene, but these were not available for use at the time of this study.
E2 ubiquitin-conjugating enzyme putative reduction-of-function, missense mutation, backcrossed 5 times
Thompson O
et al.
2013
Acknowledgments
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Funding
Partial funding was provided by a Research and Creative Opportunities Grant for Undergraduate Students from the Office of Research & Sponsored Programs at Western Washington University.
BaysNWGardnerRGSeeligLPJoazeiroCAHamptonRY200111Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation.311465-73922429}10.1038/3505052411146622BrockiePJMellemJEHillsTMadsenDMMaricqAV2001830The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated currents that regulate reversal frequency during locomotion.3140896-6273617630}10.1016/s0896-6273(01)00394-411545720BurrMLCanoFSvobodovaSBoyleLHBonameJMLehnerPJ2011118HRD1 and UBE2J1 target misfolded MHC class I heavy chains for endoplasmic reticulum-associated degradation.10850027-842420342039}10.1073/pnas.101622910821245296CookBWShawGS2012715Architecture of the catalytic HPN motif is conserved in all E2 conjugating enzymes.44520264-6021167174}10.1042/BJ2012050422563859HartACSimsSKaplanJM1995112Synaptic code for sensory modalities revealed by C. elegans GLR-1 glutamate receptor.37865520028-08368285}10.1038/378082a07477294JonesDCroweEStevensTACandidoEP20011212Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins.311474-7596RESEARCH0002RESEARCH000210.1186/gb-2001-3-1-research000211806825KowalskiJRDahlbergCLJuoP2011126The deubiquitinating enzyme USP-46 negatively regulates the degradation of glutamate receptors to control their abundance in the ventral nerve cord of Caenorhabditis elegans.3140270-647413411354}10.1523/JNEUROSCI.4765-10.201121273419SasagawaYYamanakaKOguraT200791ER E3 ubiquitin ligase HRD-1 and its specific partner chaperone BiP play important roles in ERAD and developmental growth in Caenorhabditis elegans.1291356-959710631073}10.1111/j.1365-2443.2007.01108.x17825049StewartMDRitterhoffTKlevitREBrzovicPS2016322E2 enzymes: more than just middle men.2641001-0602423440}10.1038/cr.2016.3527002219ThompsonOEdgleyMStrasbourgerPFlibotteSEwingBAdairRAuVChaudhryIFernandoLHutterHKiefferALauJLeeNMillerARaymantGShenBShendureJTaylorJTurnerEHHillierLWMoermanDGWaterstonRH2013625The million mutation project: a new approach to genetics in Caenorhabditis elegans.23101088-905117491762}10.1101/gr.157651.11323800452VembarSSBrodskyJL20081112One step at a time: endoplasmic reticulum-associated degradation.9121471-0072944957}10.1038/nrm254619002207WeberACohenIPoppODittmarGReissYSommerTRavidTJaroschE2016825Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.6351097-2765827839}10.1016/j.molcel.2016.07.02027570077WuPYHanlonMEddinsMTsuiCRogersRSJensenJPMatunisMJWeissmanAMWolbergerCPickartCM2003101A conserved catalytic residue in the ubiquitin-conjugating enzyme family.22190261-418952415250}10.1093/emboj/cdg50114517261ZhengYBrockiePJMellemJEMadsenDMMaricqAV1999101Neuronal control of locomotion in C. elegans is modified by a dominant mutation in the GLR-1 ionotropic glutamate receptor.2420896-6273347361}10.1016/s0896-6273(00)80849-110571229