Allele-specific suppression in Caenorhabditis elegans reveals details of EMS mutagenesis and a possible moonlighting interaction between the vesicular acetylcholine transporter and ERD2 receptors

Abstract

A missense mutant, unc-17(e245), which affects the Caenorhabditis elegans vesicular acetylcholine transporter UNC-17, has a severe uncoordinated phenotype, allowing efficient selection of dominant suppressors that revert this phenotype to wild-type. Such selections permitted isolation of numerous suppressors after EMS (ethyl methanesulfonate) mutagenesis, leading to demonstration of delays in mutation fixation after initial EMS treatment, as has been shown in T4 bacteriophage but not previously in eukaryotes. Three strong dominant extragenic suppressor loci have been defined, all of which act specifically on allele e245, which causes a G347R mutation in UNC-17. Two of the suppressors (sup-1 and sup-8/snb-1) have previously been shown to encode synaptic proteins able to interact directly with UNC-17. We found that the remaining suppressor, sup-2, corresponds to a mutation in erd-2.1, which encodes an endoplasmic reticulum retention protein; sup-2 causes a V186E missense mutation in transmembrane helix 7 of ERD-2.1. The same missense change introduced into the redundant paralogous gene erd-2.2 also suppressed unc-17(e245). Suppression presumably occurred by compensatory charge interactions between transmembrane helices of UNC-17 and ERD-2.1 or ERD-2.2, as previously proposed in work on suppression by SUP-1(G84E) or SUP-8(I97D)/synaptobrevin. erd-2.1(V186E) homozygotes were fully viable, but erd-2.1(V186E); erd-2.2(RNAi) exhibited synthetic lethality [like erd-2.1(RNAi); erd-2.2(RNAi)], indicating that the missense change in ERD-2.1 impairs its normal function in the secretory pathway but may allow it to adopt a novel moonlighting function as an unc-17 suppressor.

Keywords: Caenorhabditis elegans; EMS mutagenesis; KDEL receptor; acetylcholine transporter; genetic suppression; moonlighting; protein interaction; synapse.

Figure 1

Wild-type, uncoordinated, and suppressed phenotypes. Images of mixed-stage C. elegans late exponential populations of the indicated genotypes. Scale bar ∼0.5 mm.

Figure 2

Relative mobility: crawling on solid media. Procedural details are in the Materials and Methods section. e245 is an unc-17 allele, e995 is a sup-1 allele, e997 is a sup-2/erd-2.1 allele, and e1563 is a sup-8/snb-1 allele. Suppressing transgenes are abbreviated as eEx[erd-2.1*] and eEx[erd-2.2*]; the asterisks indicate that the encoded proteins carry the V186E amino acid substitution. Filled black circles represent the distances traveled by each animal, and error bars indicate the standard deviations for each set of 10 measured values. Confidence limits for the data sets are indicated by the squares and triangles over each data set, as follows. Squares designate P-values comparing a given data set with the 10-min e245 data set (open circle)—filled squares indicate P > 0.1, unfilled squares indicate P < 0.0005. Triangles designate P-values comparing a given data set with the N2 data set #2 (open circle)—filled triangles indicate P > 0.2, partially filled triangles indicate P < 0.005, unfilled triangles indicate P < 0.0005. Statistical significance was determined using the two-sample Mann-Whitney U-test.

Figure 3

Models for suppressive interaction. Diagrams showing presumed compensatory interactions between transmembrane helices of UNC-17 and suppressor proteins. TM7 sequences are almost identical in ERD-2.1 (PISVVAGIVQTVLYADFFYLYIT) and ERD-2.2 (PIVVVAGIVQTVLYADFFYLYVT). The curved arrows in each panel represent the direction of translation of each protein; note that the SUP-8/SNB-1 transmembrane helix is oriented parallel to UNC-17 TM9, whereas the interacting helices of SUP-1 and SUP-2/ERD-2.1 are antiparallel to UNC-17 TM9.

Figure 4

Suppressors of unc-17(e245) increase the abundance of UNC-17 protein. Upper panel: diagram of the anterior nervous system of a young C. elegans adult, modified from Rand et al. (2000). The rectangle with the dashed white line indicates the region of the worm included in the lower images. Panels (A–H) are immunofluorescence images of the nerve ring and ventral and dorsal nerve cords stained with a specific anti-UNC-17 monoclonal antibody (Duerr et al. 2008). Because UNC-17 is a synaptic vesicle protein, the immunostaining reflects the locations of cholinergic synapses. Representative examples of animals of each indicated genotype are shown. All nematodes were stained at the same time in the same solutions and imaged on the same day with identical “blind” image collection conditions. Relative intensity in groups of matched strains was evaluated blind 3–5 times by eye and 3 times using confocal microscopy, as described in Materials and Methods section. Anterior is to the left and ventral is down; scale bar is 20 μm.

Figure 5

RNAi knockdowns of erd-2.1 and erd-2.2 demonstrate synthetic lethality. Images show F₁ and F₂ progeny of single animals grown on dsRNA-expressing bacteria, with genotypes as indicated.