Figure 1.
odr-8 mutants are defective in Ufm1 specific protease 2.
(A) The UfSP2 gene corresponds to ORF F38A5.1. Shown are the exon/intron structure of the gene and the location of the Cysteine (blue), Histidine (green) and Aspartate (pink) boxes containing catalytic residues in the papain fold. The location of stop codons associated with the db866, ky173 and ky31 mutations is also indicated. (B–D). A transgene carrying F38A5.1a genomic fragment rescues odr-8 defects in olfaction (B), aggregation (C), and responses to changes in O2 (D). **, p<0.01 compared to non-transgenic controls (ANOVA, Bonferroni's multiple comparisons test).
Figure 2.
ODR-8 is co-expressed with ODR-4 in chemosensory neurons.
(A) ODR-4 and ODR-8 are co-expressed in the same head and tail neurons. (B–D) Cell-specific rescue of odr-8(ky31) phenotypes. Like ODR-4, ODR-8 is required in AWA neurons for odortaxis to diacetyl (B), and in ADL neurons to sustain aggregation behavior (C) and high locomotory activity in 21% oxygen (D). The podr-10 promoter drives expression only in the AWA neurons; psrh-220 drives expression in ADL neurons; podr-3 drives expression in AWA, AWB, AWC, ASH and ADF. ** p<0.01 (ANOVA, Bonferroni's multiple comparisons test) compared to non-transgenic controls.
Figure 3.
ODR-8 is required for the GPCR trafficking to the AWA cilia.
(A) Schematic drawing of the C. elegans head with the arrow indicating the AWA cell body and the arrowhead pointing to the AWA cilia. (B) ODR-8 is required to localize GFP-tagged STR-112 and STR-113 odorant receptors to AWA cilia. Cilia localization in wild type and odr-8 mutants is quantified to the right of the panel. (C) ODR-8 acts cell-autonomously to permit localization of ODR-10-GFP to the cilia of the AWA neurons. **, p<0.01 (ANOVA, Bonferroni's multiple comparisons test).
Figure 4.
ODR-10-GFP is retained in the ER in odr-8 mutants.
(A) ODR-10-GFP co-localizes predominantly with RAB-8 and LMP-1 markers in wild type animals, but with ER markers in odr-8(ky31) mutants. (B–C) ODR-4 co-localizes with the ER marker TRAM-1 (B), and ODR-4 and ODR-8 partially co-localize with each other (C).
Figure 5.
ODR-8 acts in an early step in ODR-10 trafficking to the AWA cilia.
(A) Localization of ODR-10-GFP in AWA neurons of odr-8(ky31), unc-101(m1) and odr-8(ky31); unc-101(m1) mutants. Thresholded images were obtained as described in the methods. (B) Quantitative analysis of ODR-10 and TRAM-1 co-localization in AWA neurons of odr-8(ky31), unc-101(m1) and odr-8(ky31); unc-101(m1) mutants. (C). Localization of ODR-10-GFP in AWA neurons in rab-2 mutants. Arrowheads indicate the AWA cilia, white arrows point to the AWA cell body and red arrows show the AWA processes in the nerve ring. In rab-2(n777) mutants, 23 out of 29 animals displayed normal ODR-10-GFP localization in AWA cilia, and 6 out of 29 show the ODR-10-GFP localization defects in AWA cilia. (D–E). Quantification of ODR-10-GFP signals in the cilia (D) and axons (including cell body) (E) of AWA neurons. **, p<0.01 (ANOVA, Bonferroni's multiple comparisons test); for E, rab-2(n777) shows p<0.01 compared to all other genotypes plotted.
Figure 6.
Deleting ufm-1 does not alter ODR-10 localization.
(A) Molecular nature of ufm-1(gk379) deletion allele. The gk379 deletion disrupts two adjacent operons – one comprising ufm-1 and tomm-7 and one including coq-5 and tag-307. gk379 homozygous animals die as embryos or young larvae. To score the ODR-10-GFP localization in adult animals lacking ufm-1 we rescued the gk379 lethal phenotype using a 9 kb genomic fragment in which the ufm-1 open reading frame is interrupted by a stop codon or by a combination of a stop codon and a frameshift mutation, as indicated. (B) Disrupting ufm-1 does not change the ODR-10-GFP phenotype either in wild type animals or odr-8(ky31) mutants. (C) Mutations generated by CRISPR-Cas in the ufm-1 gene. PAM refers to the protospacer adjacent motif. (D–E) Mutations in ufm-1 do not block ODR-10 trafficking to AWA cilia in wild type animals, or rescue the ODR-10 localization defect in an odr-8 mutant background. The ODR-10 cilia localization in (D) was quantified in (E).
Figure 7.
Non-catalytic action of ODR-8 Ufm1-specific protease 2.
(A) Conservation of Cysteine and Histidine boxes in UfSP2 from worm, mouse and man. The catalytic cysteine and histidine residues are marked by an arrowhead. (B–D) odr-8 transgenes in which the catalytic cysteine or histidine residues are mutated retain the ability to rescue odr-8(ky31) mutant phenotypes for odorant responses to diacetyl and benzaldehyde (B), oxygen responses (C) and biogenesis and localization of ODR-10 GPCR in AWA neurons (D). In D, ODR-10 localization in AWA cilia was scored using a scale from 0 to 4, where 0 reflects no observable localization and 4 reflects strong ODR-10 accumulation. Representative images for each genotype are shown. The odr-8 allele rescued in these assays was odr-8(ky31). ** indicates p<0.01 (ANOVA, Bonferroni's multiple comparisons test).
Figure 8.
ODR-4, ODR-8 and ODR-10 form a complex at the ER.
(A) HEK293 cells were co-transfected with ODR-4a-Flag, ODR-4b-FLAG and HA-ODR-8, and cell lysates were subjected to immunoprecipitation using indicated antibodies. The resulting precipitates were examined by immunoblot analysis with mouse anti-FLAG and mouse anti-HA antibodies. The asterisk indicates a non-specific band. (B) HEK293 cells were co-transfected with ODR-10-GFP with or without ODR-4b-FLAG, and cells lysates were subjected to immunoprecipitation using mouse anti-HA and rabbit anti-GFP antibodies. (C) HEK293 cells were co-transfected with human ODR4-FLAG and human HA-UfSP2, and cell lysates were subjected to immunoprecipitation using a FLAG antibody. The resulting precipitates were examined by immunoblot analysis with mouse anti-FLAG and mouse anti-HA antibodies. (D) HeLa cells were transiently transfected with ODR-4b-FLAG for 3 days. After fixation, the cells were subjected to immunocytochemistry using mouse anti-Flag and rabbit anti-TRAPα antibodies. (E) HeLa cells co-transfected with HA-ODR-8 and ODR-4b-FLAG as in panel D. fixed, and stained using rabbit anti-Flag and mouse anti-HA antibodies. Bar, 5 µm. (F) HeLa cells were transiently transfected with HA-ODR-8 alone or with HA-ODR-8 and ODR-4b-FLAG and cultivated for 3 days. In the panel marked ‘semi-permeabilized’ cells were treated with digitonin for 5 min prior to fixation, whereas in the panel labeled ‘intact’ the digitonin in step was omitted. Immunocytochemistry was performed using rabbit anti-Flag and mouse anti-HA antibodies.