Interdependence of the peroxisome-targeting receptors in Arabidopsis thaliana: PEX7 facilitates PEX5 accumulation and import of PTS1 cargo into peroxisomes

Abstract

Peroxisomes compartmentalize certain metabolic reactions critical to plant and animal development. The import of proteins from the cytosol into the organelle matrix depends on more than a dozen peroxin (PEX) proteins, with PEX5 and PEX7 serving as receptors that shuttle proteins bearing one of two peroxisome-targeting signals (PTSs) into the organelle. PEX5 is the PTS1 receptor; PEX7 is the PTS2 receptor. In plants and mammals, PEX7 depends on PEX5 binding to deliver PTS2 cargo into the peroxisome. In this study, we characterized a pex7 missense mutation, pex7-2, that disrupts both PEX7 cargo binding and PEX7-PEX5 interactions in yeast, as well as PEX7 protein accumulation in plants. We examined localization of peroxisomally targeted green fluorescent protein derivatives in light-grown pex7 mutants and observed not only the expected defects in PTS2 protein import but also defects in PTS1 import. These PTS1 import defects were accompanied by reduced PEX5 accumulation in light-grown pex7 seedlings. Our data suggest that PEX5 and PTS1 import depend on the PTS2 receptor PEX7 in Arabidopsis and that the environment may influence this dependence. These data advance our understanding of the biogenesis of these essential organelles and provide a possible rationale for the retention of the PTS2 pathway in some organisms.

Figure 1.

Positional cloning of the gene defective in pex7-2. (A) Recombination mapping localized the defect between F28N24 and F6N18. This interval includes the PEX7 gene (At1g29260), which encodes the PTS2 receptor. The pex7-2 C-to-T mutation causes a Thr-to-Ile missense mutation in the second WD-40 repeat; the triangle shows the position of the pex7-1 T-DNA insertion in the 5′-UTR (Woodward and Bartel, 2005). (B) pex7-2 fails to complement pex7-1. Four 8-d-old seedlings representing the range of root lengths after growth under yellow-filtered light on medium without or with 10 μM IBA are shown. Col-0 plants were used as wild type (PEX7/PEX7), and the mean percentage of root length on IBA versus unsupplemented medium is shown; n ≥ 10. Bar, 1 cm. (C) 35S-PEX7a rescues pex7-2 sucrose dependence. Seedlings were grown under white light for 1 d and transferred to the dark for five additional days on medium with or without 0.5% sucrose. Bars show mean hypocotyl lengths + SE; n ≥ 9. (D) 35S-PEX7a restores pex7-2 IBA responsiveness. Bars show mean root lengths (+SE; n ≥ 10) of 8-d-old seedlings grown under yellow-filtered light on 10 μM IBA or medium containing no hormone.

Figure 2.

pex7-2 IBA resistance and sucrose dependence suggest IBA and fatty acid β-oxidation defects. (A) pex mutants are resistant to root elongation inhibition by IBA. Seeds were stratified 3 d at 4°C before 8 d of growth under yellow-filtered light on medium without hormone or supplemented with IBA. Bars show mean root lengths + SE; n ≥ 8. (B) pex mutants are resistant to lateral root promotion by IBA. Four-day-old seedlings grown under yellow-filtered light were transferred from hormone-free medium to medium containing IBA, the synthetic auxin NAA, or no hormone. After four additional days, root length was measured (top) and lateral roots emerged from the primary root were counted (bottom). Bars show means + SE; n ≥ 10. (C) Sucrose dependence of pex mutants. Seedlings were grown with or without sucrose for 1 d in light and 5 d in darkness (top) or for 8 d under white light (bottom). Bars show mean hypocotyl or root lengths + SE; n ≥ 9.

Figure 3.

Developmental defects of pex5 and pex7 mutant plants. (A) pex5-10, pex7-2, and pex5 pex7-1 double mutants are developmentally delayed. Seedlings were grown for 26 d on medium supplemented with 0.5% sucrose under white light. Bar, 1 cm. (B) pex5 and pex7 mutants produce relatively healthy and fertile adult plants. Plants from A were transferred to soil and grown under constant white light. Representative 55-d-old plants are shown. Bar, 10 cm. (C) pex7-2 confers embryo lethality in combination with pex5 mutants. Valves were removed from siliques of the indicated genotypes to reveal developing seeds. Images are arranged in order of increasing silique maturity (top to bottom). (D) Mature seed phenotypes of lines in panel C; the fraction of shrunken versus filled seeds is shown below the images. Bar, 1 mm.

Figure 4.

The pex7-2 lesion disrupts PEX5 and PTS2-cargo binding in yeast. (A) Yeast two-hybrid assays were conducted with PEX7 and pex7-2 fused to the GAL4-DNA binding domain (BD) and PEX5, PED1 (a PTS2 protein), and tPED1 (without the PTS2 region) fused to the GAL4 activation domain (AD). Serial dilutions spotted onto permissive plates (+His) and plates on which growth requires interaction (−His) are shown after incubation at 30°C for 2 d. (B) Proteins extracted from yeast strains in A were separated using SDS-polyacrylamide gel electrophoresis and immunoblots were probed with the indicated antibodies. Extracts from 8-d-old wild-type Arabidopsis seedlings and the untransformed AH109 yeast were included as controls.

Figure 5.

Protein accumulation in pex7 and pex5 single and double mutants. (A and B) Light-grown pex7 mutants have PTS2 processing defects (A) and reduced PEX5 accumulation (B). Proteins extracted from 7-d-old seedlings grown on 0.5% sucrose under white light were analyzed by immunoblotting with the indicated antibodies. (C and D) Dark-grown pex7 mutants have PTS2 processing defects (C) and less severe receptor accumulation defects (D). Proteins were extracted from seedlings grown 1 d in light and 4 d in darkness on 0.5% sucrose and analyzed by immunoblotting. Protein was normalized to the same volume.

Figure 6.

pex7-2 defects in matrix protein import. (A) Cotyledons of 7-d-old light-grown seedlings or of seedlings grown 1 d in light and 4 d in darkness on 0.5% sucrose were analyzed using confocal fluorescence microscopy. PTS2 protein targeting was visualized by imaging fluorescence from a GFP derivative (PTS2-GFP) carrying the N-terminal peroxisome targeting signal from PED1 (Woodward and Bartel, 2005) in epidermal cells (first column) and the underlying mesophyll cells (second column). The mesophyll column shows merged images of GFP (green) and chlorophyll (chl; magenta) fluorescence. The large central vacuole of expanded plant cells consolidates most of the cytosol at the cell margins. Dark-grown cotyledons were briefly stained with propidium iodide, which stains cell walls, before visualization of epidermal cells; the last column shows merged image of GFP (green; third column) and propidium iodide (magenta) fluorescence. Bar, 20 μm. (B) Cotyledons of seedlings grown as described in A were analyzed as described in A. PTS1 protein targeting was visualized by imaging fluorescence from a GFP derivative (GFP-PTS1) carrying a C-terminal PTS1 (Zolman and Bartel, 2004). Bar, 20 μm.