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First published online June 27, 2005
doi: 10.1242/10.1242/dev.01909

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Multidrug resistance-associated protein MRP-1 regulates dauer diapause by its export activity in Caenorhabditis elegans

Tomoko Yabe^1,*, Norio Suzuki^1,, Tatsuhiko Furukawa², Takeshi Ishihara^1, and Isao Katsura^1,

¹ Structural Biology Center, National Institute of Genetics, and Department of Genetics, The Graduate University for Advanced Studies, Mishima 411-8540, Japan
² Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8544, Japan

View larger version (25K): [in a new window]   Fig. 1. Dauer larva formation of unc-31(e169);sdf-14 double mutants. All the unc-31(e169);sdf-14 double mutants formed dauer larvae at high percentages at 25.5°C, whereas the unc-31(e169) single mutant formed dauer larvae only at 27°C and not at 25.5°C. sdf-14 mutants and wild-type animals did not form dauer larvae at either 25.5°C or 27°C. The results of the deletion mutant mrp-1(pk89) and unc-31(e169);mrp-1(pk89) are also shown, because sdf-14 was found to be allelic to mrp-1 in this study. The means of three plates are shown (33-199 animals/plate). The error bars indicate s.e.m. in all the figures of this paper.

View larger version (37K): [in a new window]   Fig. 2. C. elegans MRP-1. (A) Putative topology of the C. elegans MRP-1 protein (top); structure of the mrp-1 gene and its cDNAs (bottom). Like human MRP1, C. elegans MRP-1 seems to have two nucleotide-binding domains (NBDs) and three membrane-spanning domains (MSDs). The mrp-1 gene has 20 exons, of which the thirteenth exon is variable. The mutation sites of ut151, ut153 and ut155, as well as the region deleted in pk89 are shown in the gene structure. The C. elegans database WormBase listed the mrp-1 cDNA isoforms, a-, b- and c-types, as well as d.1-, d.2- and d.3-types. The latter three cDNAs are not shown in this figure because they encode only short polypeptides. We found a fifth isoform (e-type), but could not confirm the existence of the a-type. (B) Constructs used for transgene experiments.

View larger version (38K): [in a new window]   Fig. 3. Effect of sodium arsenite on the dauer larva formation of various strains. (A-C) Sodium arsenite did not enhance the dauer larva formation of mrp-1(ut153) (A), but enhanced that of unc-31(e169) (B) and unc-31(e169);mrp-1(ut153) (C). (D) The effects of sodium arsenite on unc-31(e169) and unc-31(e169);mrp-1(ut153) were measured at the same temperature for comparison. The means of three plates are shown (53-229 animals/plate).

View larger version (23K): [in a new window]   Fig. 4. Functional difference among the mrp-1 isoforms. The b- and c-type isoforms, but not the e-type isoform, can rescue of the Daf-c phenotype of unc-31(e169);mrp-1(ut153). The means of multiple lines are shown (two lines of b-type, eight lines of c-type, and four lines of e-type). The assays were carried out with 13-128 animals/plate at 25.5°C. Animals carrying an extrachromosomal array of transgenes segregated those that had lost the extrachomosomal array. Dauer formation of these animals was also examined and is shown as a control on the left of each data set.

View larger version (60K): [in a new window]   Fig. 5. Expression pattern of MRP-1. (A) Dauer larva formation of unc-31(e169);mrp-1(ut153) was rescued by the mrp-1::GFP fusion gene, showing that it was functional. The means of four plates are shown (21-94 animals/plate). (B) The mrp-1::GFP fusion gene was expressed in various tissues (L1 larva). (C) Expression in seam cells (L2 or L3 larva). (D) Neuronal expression; neurons are indicated by arrowheads. (E) Localization of GFP at the cell membrane. The expression patterns were observed with wild-type animals.

View larger version (34K): [in a new window]   Fig. 6. MRP-1 needs to be expressed in multiple tissues for the wild-type phenotype. Functional mrp-1::GFP fusion genes driven by various tissue-specific promoters (myo-2 promoter for pharyngeal muscles, ges-1 promoter for intestinal cells, and H20 promoter for neurons) were introduced either separately or in combination into unc-31(e169);mrp-1(ut153) double mutant animals. The results show that expression in multiple tissues is necessary to rescue the abnormality of dauer larva formation efficiently. The means of two to eight lines are shown (19-92 animals/line). Because the expression in neurons and intestinal cells looked weaker than in pharyngeal cells, we increased the concentration of the former two DNA constructs by 4-fold (40 ng/µl). However, expression in one tissue still resulted in partial rescue. Animals carrying an extrachromosomal array of transgenes segregated those that had lost the extrachromosomal array. Dauer formation of these animals was also examined and is shown as a control on the left of each data set.

View larger version (18K): [in a new window]   Fig. 7. Human MRP1 can substitute for C. elegans MRP-1 in dauer larva regulation. (A) The dauer larva formation of the unc-31(e169);mrp-1(ut153) double mutant was suppressed efficiently by an extrachromosomal array of the wild-type human MRP1 cDNA driven by the C. elegans mrp-1 promoter. (B) The suppression of dauer larva formation by human MRP1 cDNA was partially antagonized by the human MRP1 inhibitor PAK-104P. (C) The suppression of dauer larva formation by C. elegans mrp-1 genomic DNA was not antagonized by PAK-104P. The means of three plates (42-107 animals/plate) are shown. As PAK-104P was dissolved in DMSO, the final concentration of DMSO is shown in the figure. Animals carrying an extrachromosomal array of transgenes segregated those that had lost the extrachromosomal array. Dauer formation of these animals was also examined and is shown as a control on the left of each data set.

View larger version (29K): [in a new window]   Fig. 8. The mrp-1 gene acts neither in the cGMP nor in the TGF-ß signaling pathway for dauer larva regulation. (A) The mrp-1(pk89) mutation enhanced the dauer larva formation of daf-11(m47) (cGMP signaling pathway) and daf-1(m40) (TGF-ß signaling pathway) to a small and similar extent. By contrast, it enhanced that of daf-2(e1370ts) (insulin signaling pathway) to a large extent. (B) The dauer larva formation of daf-2(e1370);mrp-1(pk89) was not suppressed by che-3(e1124), a daf-d mutation in the cGMP signaling pathway. (C) The dauer larva formation of daf-2(e1370);mrp-1(pk89) was not suppressed by daf-5(e1386), a daf-d mutation in the TGF-ß signaling pathway. (D) As expected, the dauer larva formation of daf-2(e1370);mrp-1(pk89) was suppressed by daf-16(mu86), a daf-d mutation in the insulin signaling pathway, which suppresses daf-2(e1370).