Figure 1.
vps-331 mutations suppress
vps-45(0) ts larval arrest and lethality:(A) Genomic region of the
vps-33.1 gene. Exons are indicated by gray boxes, mutation sites in
jq28 and
syb8647 are underlined, and amino acid substitutions are indicated below. The VPS-33.1 proteins contain three domains, with domain 3 split into domain 3a and domain 3b. The suppressor mutation associated residue methionine 376 (M376I, arrowhead) is located in the domain 3a. The amino acid sequence alignments of VPS33A and VPS45 are shown below [VPS33A: Homo sapiens (NP_075067.2), Danio rerio (NP_001093443.1), Drosophila melanogaster (AAF48972), Caenorhabditis elegans (CCD61712.1), Saccharomyces cerevisiae (KZV09647.1), VPS45: Homo sapiens (NP_009190), Danio rerio (NP_001243585.1), Drosophila melanogaster (AAF54403.1), Caenorhabditis elegans (NP_741714.1), Saccharomyces cerevisiae (CAA96801.1)]. The conserved amino acids of the VPS33 or VPS45 proteins are highlighted in grey and the conserved amino acids of both proteins are highlighted in light blue. The M376 is indicated by a red box. The strictly conserved proline (proline 363 in C. elegans) in the domain 3a (Hu et al., 2011) is indicated by a blue asterisk. (B) Structure of the C. elegans VPS-33.1 protein based on I-TASSER structural modeling. The 3D model with the highest confidence score is shown. The distal tip of domain 3a which is expected to interact with SNAREs/SNARE complex (Pieren, Schmidt and Mayer, 2010; Hu et al., 2011; Baker, Jeffrey and Hughson, 2013) is shown in green. The residue M376 is indicated by a space-filling representation in red. The conserved proline, P363, is highlighted in blue. (C) Representative images of wild-type,
vps-45(0),
vps-33.1(
jq28);
vps-45(0),
vps-33.1(
syb8647);
vps-45(0),
vps-33.1(
syb8647) animals grown from eggs at 25°C for 76 h.
vps-45(0) represents the null allele
vps-45(
tm246). Scale bars, 200 µm. (D) Quantification of body length of animals in (C) (n=23-29). mean±SD. Statistics: one-way ANOVA with Dunnet's post hoc test. ****p<0.0001. ns: not significant. (E) Percentage of adults in the animals measured in (D) (n=23-29). ***p<0.001. ns: not significant. Statistics: Fisher's exact test, two-tailed.