The Arabidopsis peroxisomal targeting signal type 2 receptor PEX7 is necessary for peroxisome function and dependent on PEX5

Abstract

Plant peroxisomal proteins catalyze key metabolic reactions. Several peroxisome biogenesis PEROXIN (PEX) genes encode proteins acting in the import of targeted proteins necessary for these processes into the peroxisomal matrix. Most peroxisomal matrix proteins bear characterized Peroxisomal Targeting Signals (PTS1 or PTS2), which are bound by the receptors PEX5 or PEX7, respectively, for import into peroxisomes. Here we describe the isolation and characterization of an Arabidopsis peroxin mutant, pex7-1, which displays peroxisome-defective phenotypes including reduced PTS2 protein import. We also demonstrate that the pex5-1 PTS1 receptor mutant, which contains a lesion in a domain conserved among PEX7-binding proteins from various organisms, is defective not in PTS1 protein import, but rather in PTS2 protein import. Combining these mutations in a pex7-1 pex5-1 double mutant abolishes detectable PTS2 protein import and yields seedlings that are entirely sucrose-dependent for establishment, suggesting a severe block in peroxisomal fatty acid beta-oxidation. Adult pex7-1 pex5-1 plants have reduced stature and bear abnormally shaped seeds, few of which are viable. The pex7-1 pex5-1 seedlings that germinate have dramatically fewer lateral roots and often display fused cotyledons, phenotypes associated with reduced auxin response. Thus PTS2-directed peroxisomal import is necessary for normal embryonic development, seedling establishment, and vegetative growth.

Figure 1.

Organization of Arabidopsis PEX7 and PEX5 proteins. (A) Schematic showing domain architecture of Arabidopsis PEX7 and PEX5 proteins. The bracket above PEX5 is the region shown in C, and the asterisk indicates the Ser to Leu mutation indicated in C. (B) PEX7 is the single Arabidopsis (At) homolog of human (Hs) PEX7 and is highly similar to tomato (Le) GenBank accession AY186749 and rice (Os) TIGR gene temporary ID 8351.t01315 (OsPEX7). (C) Partial alignment of Arabidopsis PEX5 with orthologues from rice, human, yeast (Sc), and roundworm (Ce). pex5-1 contains a missense mutation in a conserved serine (Zolman et al., 2000) needed for PEX7 interaction and PTS2-containing protein import into peroxisomes in Chinese hamster (Matsumura et al., 2000). Note the absence of this domain in CePEX5; C. elegans does not contain PEX7 or a PTS2 import pathway (Gurvitz et al., 2000; Motley et al., 2000). Sequences were aligned with the MegAlign program (DNA Star, Madison, WI) using the ClustalW method; residues identical in three or more sequences are shaded black, residues chemically similar in three or more sequences are shaded gray. WD-40 domains determined by Pfam (Bateman et al., 2002); PPR and TPR domains after Zolman et al. (2000).

Figure 2.

pex7-1, pex5-1, and pex7-1 pex5-1 are deficient in peroxisomal processes. (A) Root elongation on IBA. Root lengths of 8-d-old plants grown on sucrose-supplemented medium with or without addition of 5 μM IBA under yellow light filters are shown. (B) Percent root elongation on 5 μM IBA versus hormone-free medium. Plants were grown as in A. (C) Lateral root number. Lateral roots of 9-d-old plants grown on PNS under yellow light filters were counted under a dissecting microscope. (D) Hypocotyl elongation in darkness with and without sucrose. Plants were grown one day in white light and then were grown in darkness for 5 additional days. Bars, means + SDs; n ≥ 6 in A, n ≥ 10 in B-D. ^*Significantly different from wild type (p < 0.01; one-tailed t test assuming unequal variance). (E) pex7-1 pex5-1 does not develop after germination without sucrose in the light. Wild-type (left) or pex7-1 pex5-1 (right) plants were grown for 7 d under white light on medium lacking sucrose. Scale bars, 1 mm.

Figure 3.

pex7-1 and pex5-1 are deficient in PTS2 protein import into peroxisomes. Three-day-old root tips from seedlings grown in 0.1% agar in white light (left panels) and 7-d-old root hair cells (right panels) are shown for wild-type, pex7-1, and pex5-1, and transformants expressing PTS1-GFP, PTS2-GFP, and cytoplasmic GFP. Note the punctate fluorescence in plants expressing PTS1-GFP and wild-type plants expressing PTS2-GFP; fluorescence is diffuse in pex7-1 and pex5-1 expressing PTS2-GFP, indicating disrupted PTS2 protein import into peroxisomes. Untransformed plant images were captured at the maximum exposure time used for any of the transformed lines. Scale bars, 200 μm.

Figure 4.

pex7-1 and pex5-1 are deficient in import of the PTS2 protein thiolase into peroxisomes. (A) Thiolase import defects. Precursor thiolase protein is translated with an N-terminal, PTS2-containing sequence that is cleaved after entry into peroxisomes to produce mature thiolase. Protein was extracted from seedlings grown on sucrose and visualized using an antithiolase antibody (Kato et al., 1996). (B) Thiolase import defects are exaggerated in plants grown without exogenous sucrose. pex7-1 pex5-1 was omitted because of developmental arrest in the absence of sucrose. (C) PEX5 protein is present in all mutants. Protein was extracted from seedlings grown on sucrose and visualized using anti-PEX5 antibody (Zolman and Bartel, 2004). Positions of molecular mass markers (in kDa) are indicated at the left in A-C. (D) PEX7 message is present in all mutants. PEX7 mRNA levels relative to an APRT control in wild type and each mutant were determined using quantitative real-time reverse-transcription PCR. Error bars, SDs of mean PEX7 levels expressed in arbitrary units.

Figure 5.

pex7-1 pex5-1 double mutant phenotypes. (A) Seed development is aberrant in pex7-1 pex5-1. One valve was removed from wild-type and pex7-1 pex5-1 siliques to reveal developing seeds of increasing age from left to right. Scale bar, 1 mm. (B) Mature pex7-1 pex5-1 seeds are shrunken. Scale bar, 1 mm. (C) pex7-1 pex5-1 adult plants have reduced stature. Plants were grown under white light on PNS for 14 d, then transferred to soil, and grown an additional 17 d. Scale bar, 1 cm. (D) Percent seeds with normal filled morphology. Mature seeds were assayed for plump appearance. Bars, means + SDs from progeny of three plants of the indicated genotype; n ≥ 60 seeds per plant. (E) Percent germination in single and double mutants. pex7-1 pex5-1 were sorted by seed morphology in D. Seedlings were grown on medium supplemented with 45 mM sucrose and assayed for germination (radicle emergence) after 9 d. Bars, means + SDs (or SE of measurement for pex5-1) from progeny of three plants of the indicated genotype (progeny of two pex5-1 plants); n ≥ 23 seeds per plant, except pex7-1 pex5-1 filled seeds where n ≥ 10 seeds per plant. ^*Significantly different from wild type (p < 0.02; one-tailed t test assuming unequal variance).

Figure 6.

Cotyledon fusion in the pex7-1 pex5-1 double mutant. pex7-1 pex5-1 plants were grown for 7 d on sucrose-supplemented medium and frequencies of plants with (A) wild-type, (B) asymmetric, (C) partially fused, and (D) fused cotyledons were determined. Ratios below B, C, and D represent fraction of pex7-1 pex5-1 with the indicated degree of fusion among progeny of three double mutant plants. Scale bar, 1 mm.

Figure 7.

Model for PTS2 protein import into plant peroxisomes. (A) PEX7 binds both PTS2 protein cargo and the PTS1 protein receptor PEX5. Delivery of PTS2 protein into the peroxisome is dependent on complex formation with PEX5. PTS2 protein cargo is necessary for peroxisomal processes of fatty acid β-oxidation and conversion of IBA into the active auxin IAA. (B) Functional PEX7 may be expressed at reduced levels in the pex7-1 mutant, resulting in reduced PTS2 protein import and IBA resistance. (C) PEX7 binding with pex5-1 is inefficient, resulting in decreased PTS2 protein import and IBA resistance. (D) Two partial defects are combined in pex7-1 pex5-1 to nearly eliminate PTS2 protein import, resulting in severe developmental defects.