Peroxisomal ubiquitin-protein ligases peroxin2 and peroxin10 have distinct but synergistic roles in matrix protein import and peroxin5 retrotranslocation in Arabidopsis

Abstract

Peroxisomal matrix proteins carry peroxisomal targeting signals (PTSs), PTS1 or PTS2, and are imported into the organelle with the assistance of peroxin (PEX) proteins. From a microscopy-based screen to identify Arabidopsis (Arabidopsis thaliana) mutants defective in matrix protein degradation, we isolated unique mutations in PEX2 and PEX10, which encode ubiquitin-protein ligases anchored in the peroxisomal membrane. In yeast (Saccharomyces cerevisiae), PEX2, PEX10, and a third ligase, PEX12, ubiquitinate a peroxisome matrix protein receptor, PEX5, allowing the PEX1 and PEX6 ATP-hydrolyzing enzymes to retrotranslocate PEX5 out of the membrane after cargo delivery. We found that the pex2-1 and pex10-2 Arabidopsis mutants exhibited defects in peroxisomal physiology and matrix protein import. Moreover, the pex2-1 pex10-2 double mutant exhibited severely impaired growth and synergistic physiological defects, suggesting that PEX2 and PEX10 function cooperatively in the wild type. The pex2-1 lesion restored the unusually low PEX5 levels in the pex6-1 mutant, implicating PEX2 in PEX5 degradation when retrotranslocation is impaired. PEX5 overexpression altered pex10-2 but not pex2-1 defects, suggesting that PEX10 facilitates PEX5 retrotranslocation from the peroxisomal membrane. Although the pex2-1 pex10-2 double mutant displayed severe import defects of both PTS1 and PTS2 proteins into peroxisomes, both pex2-1 and pex10-2 single mutants exhibited clear import defects of PTS1 proteins but apparently normal PTS2 import. A similar PTS1-specific pattern was observed in the pex4-1 ubiquitin-conjugating enzyme mutant. Our results indicate that Arabidopsis PEX2 and PEX10 cooperate to support import of matrix proteins into plant peroxisomes and suggest that some PTS2 import can still occur when PEX5 retrotranslocation is slowed.

Figure 1.

Recombination mapping of pfl36 and pfl81 reveals mutations in PEX2 and PEX10. A, PEX proteins (numbered) implicated in matrix protein import serve as receptors (PEX5 and PEX7) recognizing PTS1 or PTS2 cargo proteins, dock receptors at the peroxisomal membrane (PEX13 and PEX14), or assist in PEX5 retrotranslocation (for review, see Hu et al., 2012). In yeast, the RING-finger proteins PEX2, PEX10, and PEX12 participate as heterooligomers in different modes of PEX5 ubiquitination (Ub; for review, see Platta et al., 2013). B, GFP-ICL fluorescence is detected in both 5- and 7-d-old pex10-2 (pfl81) seedlings carrying ICLp:GFP-ICL, whereas GFP-ICL is easily detected in 5- but not 7-d-old wild-type (Wt) ICLp:GFP-ICL seedlings. Hypocotyls of light-grown seedlings were imaged for GFP fluorescence using confocal microscopy. Bar = 20 µm. C, pfl36 was mapped to the bottom of chromosome 1 near the PEX2 gene using the phenotypes of prolonged GFP-ICL fluorescence accompanied by PMDH processing defects. The number of recombinants over the number of chromosomes scored is indicated for each marker assayed. D, A gene diagram of PEX2 depicting exons as rectangles and introns as lines. A missense mutation in the fourth exon of PEX2 in pfl36 (pex2-1) changes Arg161 to Lys. Three other pex2 alleles are indicated: pex2-2, ted3 (Hu et al., 2002), and the transfer DNA insertion allele Salk_033081 that confers embryo lethality (Hu et al., 2002). E, The locations of the lesions in viable pex2 alleles are indicated on a diagram depicting the PEX2 protein domains, which include two predicted transmembrane domains (TMDs) and a C-terminal RING domain. F, pfl81 was mapped using the IBA resistance phenotype to an interval on the lower arm of chromosome 2 that contained the PEX10 gene. The number of recombinants over the number of chromosomes scored is indicated for each marker assayed. G, pfl81 (pex10-2) carries a PEX10 splicing mutation in the last nucleotide of intron 8. Four other reported pex10 mutants are indicated on the gene diagram: the pex10-1 transfer DNA insertion allele (Schumann et al., 2003; Sparkes et al., 2003) and three Targeting Induced Local Lesions In Genomes (TILLING) alleles: pex10-G93E, pex10-P126S, and pex10-W313* (Prestele et al., 2010). H, The locations of the lesions in the two viable pex10 alleles are indicated on a diagram depicting the PEX10 protein domains, which include two predicted TMDs and a C-terminal RING domain.

Figure 2.

pex2-1 and pex10-2 mutants display IBA resistance and PTS2 processing defects. A, Root lengths of 8-d-old wild-type (Col-0) or pex seedlings grown in yellow-filtered light in the presence or absence of Suc or on Suc-supplemented medium containing 10 µm IBA are shown. Error bars show sds of the means (n ≥ 12). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). B, Hypocotyl lengths of 6-d-old wild-type (Col-0) or pex seedlings grown in the dark in the presence or absence of Suc or on Suc-supplemented medium containing 30 µm IBA are shown. Error bars show sds of the means (n ≥ 13). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). C, Lateral roots per millimeter of root length of 7-d-old wild-type (Col-0) or pex seedlings 3 d after transfer to Suc-containing medium with or without 10 µm IBA are shown. Error bars show sds of the means (n ≥ 8). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). D, Protein extracts from the 8-d-old seedlings grown in the light on 0.5% Suc from A were processed for immunoblotting. The membrane was serially probed with antibodies to the indicated proteins. The positions of molecular mass markers (in kilodaltons) are indicated on the right. PMDH is synthesized as a precursor (p) containing the PTS2 signal that is processed into the mature (m) protein in the peroxisome. HSC70 was used to monitor loading. E, Protein extracts from 4-d-old seedlings grown in the light on 0.5% Suc were processed for immunoblotting and probed with the indicated antibodies. HSC70 was used to monitor loading. Experiments in A to E were repeated at least three times with similar results. *, A cross-reacting band that appears with the PEX10 antibody.

Figure 3.

Stabilization of glyoxylate cycle enzymes in pex2 and pex10 mutants. ICL and MLS are stabilized in several pex2 and pex10 mutants. Protein extracts from 4-, 5-, and 6-d-old light-grown seedlings of the wild type (Col-0) and pex2 and pex10 alleles were processed for immunoblotting. Membranes from duplicate gels were serially probed with antibodies to the indicated proteins. PMDH is synthesized as a precursor (p) with a cleavable PTS2 signal that is processed into mature (m) PMDH in the peroxisome. HSC70 was used to monitor loading. The positions of molecular mass markers (in kilodaltons) are indicated on the left. *, A cross-reacting band that appears with the ICL antibody.

Figure 4.

PEX2 (but not PEX10) rescues pex2-1 physiological and molecular defects. A, Overexpressing PEX2 (but not PEX10) restored IBA-responsive lateral rooting to pex2-1. Four-day-old seedlings were transferred to the indicated media for 4 additional d. Error bars show sds of the mean number of lateral roots per millimeter of root length (n = 8). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). B, Expressing PEX2 (but not PEX10) rescued the PMDH PTS2 processing defect of pex2-1. Protein extracts from 8-d-old light-grown seedlings were processed for immunoblotting, and membranes from duplicate gels were serially probed with the indicated antibodies. The HA antibody detected both HA-PEX2 and HA-PEX10. The PEX2 (or PEX10) antibody detected untagged and HA-tagged PEX2 (or PEX10). HSC70 was used to monitor loading. *, A cross-reacting band that appears with the PEX2 antibody (Sparkes et al., 2005). C, The ICL and MLS stabilization of pex2-1 was rescued by transgenic HA-PEX2 (but not HA-PEX10). Extracts from 3-d-old to 5-d-old seedlings were processed for immunoblotting, and membranes from duplicate gels were probed with the indicated antibodies. *, A cross-reacting band that appears with the ICL antibody. In A and B, two independent transgenic lines (1 and 2) were examined for each transgene; C used the HA-PEX2 and HA-PEX10 lines showing higher steady-state protein levels. These experiments were repeated twice with similar results.

Figure 5.

PEX10 (but not PEX2) rescues pex10-2 physiological and molecular defects. A, Overexpressing PEX10 (but not PEX2) rescued the pex10-2 IBA resistance of dark-grown hypocotyls. Seedlings were grown on the indicated media in the dark for 5 d. Error bars show sds of the mean hypocotyl lengths (n = 12). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). B and C, The PTS2 processing defect and ICL stabilization of pex10-2 were rescued by expressing HA-PEX10. Protein extracts from light-grown seedlings were processed for immunoblotting as described in Figure 3. PEX10 levels decreased from 3 to 5 d, and pex10-2 had lower endogenous PEX10 levels, even on day 3. *, Cross-reacting bands of the ICL or PEX2 antibodies. In A and B, two independent transgenic lines (1 and 2) were examined for each transgene; C used the HA-PEX2 and HA-PEX10 lines showing higher steady-state protein levels. These experiments were repeated twice with similar results.

Figure 6.

Peroxisomally targeted PTS1 proteins are mislocalized in pex2-1 and pex10-2 seedlings. Light-grown cotyledon epidermal and mesophyll cells were imaged in 5- or 8-d-old seedlings carrying the indicated reporters using confocal microscopy. GFP-PTS1 (A and C), PTS2^PMDH1-GFP (B), and PTS2^PED1-GFP (D) fluorescence is shown in white (epidermis) or green (mesophyll), and chlorophyll fluorescence is shown in magenta (mesophyll). These experiments were repeated at least twice with similar results. Bars = 20 µm.

Figure 7.

PEX5 overexpression alters pex10-2 defects but does not notably impact pex2-1. A, Hypocotyl lengths of 7-d-old wild-type (Wt; Col-0) or pex seedlings without or with PEX5 overexpression (35S:PEX5) grown in the dark in the presence or absence of Suc or on Suc-supplemented medium containing 30 µm IBA are shown. Error bars show sds of the means (n ≥ 22). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). B, Lateral roots per millimeter of root length of 7-d-old seedlings 3 d after transfer to Suc-containing medium with or without 10 µm IBA are shown. Error bars show sds of the means (n ≥ 8). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). C, Protein extracts from 4-, 5-, and 6-d-old seedlings grown in the light on 0.5% Suc were processed for immunoblotting. Membranes from duplicate gels were serially probed with antibodies to the indicated proteins. *, A cross-reacting band that appears with the ICL antibody. The positions of molecular mass markers (in kilodaltons) are indicated on the right. PMDH and thiolase are synthesized as precursor (p) containing the PTS2 signal that is processed into the mature (m) protein in the peroxisome. HSC70 was used to monitor loading. These experiments were repeated twice with similar results.

Figure 8.

pex2-1 restores PEX5 protein levels to pex6-1 but not pex7-1. A, Root lengths of 8-d-old wild-type (Wt; Col-0) or pex seedlings grown in yellow-filtered light in the presence of Suc or on Suc-supplemented medium containing 10 µm IBA are shown. Error bars show sds of the means (n ≥ 18). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). B, Root lengths of 8-d-old seedlings grown in yellow-filtered light in the presence or absence of Suc. Error bars show sds of the means (n ≥ 16). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). C, Photographs of 8-d-old seedlings from B. Bar = 1 cm. D, Protein extracts from 8-d-old seedlings grown in the light on 0.5% Suc from A were processed for immunoblotting. The membrane was serially probed with antibodies to the indicated proteins. PMDH and thiolase are synthesized as precursors (p) containing the PTS2 signal that is processed into the mature (m) protein in the peroxisome. Numbers below the PMDH or thiolase bands indicate the percentages remaining unprocessed in each lane. HSC70 was used to monitor loading, and numbers below PEX5 and PEX7 bands indicate ratios of the band to HSC70 that are normalized such that the ratio from the wild type was set to 1.0. These experiments were repeated three times with similar results.

Figure 9.

The pex2-1 pex10-2 double mutant displays synergistic physiological and molecular defects. A, Hypocotyl lengths of 6-d-old seedlings grown in the dark in the presence or absence of Suc or on Suc-supplemented medium containing 30 µm IBA are shown. Error bars show sds of the means (n ≥ 16). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). B, Root lengths of 8-d-old seedlings grown in yellow light in the presence or absence of Suc or on Suc-supplemented medium containing 7 µm IBA are shown. Error bars show sds of the means (n = 20). Different letters above bars represent significantly different means (one-way ANOVA, P < 0.005). C, Protein extracts from the 8-d-old seedlings grown in the light on 0.5% Suc from B were processed for immunoblotting. The membrane was serially probed with antibodies to the indicated proteins. The positions of molecular mass markers (in kilodaltons) are indicated on the right. PMDH and thiolase are synthesized as a precursor (p) containing the PTS2 signal that is processed into the mature (m) protein in the peroxisome. HSC70 was used to monitor loading. D, Photograph of the 17-d-old light-grown wild type (Wt; Col-0), pex2-1, pex10-2, and the pex2-1 pex10-2 double mutant. Bar = 1 cm. E, Protein extracts from 17-d-old seedlings grown in the light on 0.5% Suc were processed for immunoblotting. The membrane was serially probed with antibodies to the indicated proteins. The positions of molecular mass markers (in kilodaltons) are indicated on the right. PMDH and thiolase are synthesized as precursors containing the PTS2 signal that is processed into the mature proteins in the peroxisome. HSC70 was used to monitor loading. F, Light-grown cotyledon epidermal and mesophyll cells were imaged in 8-d-old seedlings using confocal microscopy. GFP-PTS1 and PTS2^PMDH1-GFP fluorescence is shown in white (epidermis) or green (mesophyll); chlorophyll fluorescence is shown in magenta (mesophyll). These experiments were repeated at least twice with similar results. Bar = 20 µm.