A pex1 missense mutation improves peroxisome function in a subset of Arabidopsis pex6 mutants without restoring PEX5 recycling

Abstract

Peroxisomes are eukaryotic organelles critical for plant and human development because they house essential metabolic functions, such as fatty acid β-oxidation. The interacting ATPases PEX1 and PEX6 contribute to peroxisome function by recycling PEX5, a cytosolic receptor needed to import proteins targeted to the peroxisomal matrix. Arabidopsis pex6 mutants exhibit low PEX5 levels and defects in peroxisomal matrix protein import, oil body utilization, peroxisomal metabolism, and seedling growth. These defects are hypothesized to stem from impaired PEX5 retrotranslocation leading to PEX5 polyubiquitination and consequent degradation of PEX5 via the proteasome or of the entire organelle via autophagy. We recovered a pex1 missense mutation in a screen for second-site suppressors that restore growth to the pex6-1 mutant. Surprisingly, this pex1-1 mutation ameliorated the metabolic and physiological defects of pex6-1 without restoring PEX5 levels. Similarly, preventing autophagy by introducing an atg7-null allele partially rescued pex6-1 physiological defects without restoring PEX5 levels. atg7 synergistically improved matrix protein import in pex1-1 pex6-1, implying that pex1-1 improves peroxisome function in pex6-1 without impeding autophagy of peroxisomes (i.e., pexophagy). pex1-1 differentially improved peroxisome function in various pex6 alleles but worsened the physiological and molecular defects of a pex26 mutant, which is defective in the tether anchoring the PEX1-PEX6 hexamer to the peroxisome. Our results support the hypothesis that, beyond PEX5 recycling, PEX1 and PEX6 have additional functions in peroxisome homeostasis and perhaps in oil body utilization.

Keywords: AAA ATPase; oil bodies; peroxin; peroxisome; pexophagy.

Fig. 1.

pex1-1 identification. (A) PEX1 gene diagram showing exons (rectangles), introns (lines), and the location of pex1-1, a G-to-A transition that yields a Glu748-to-Lys substitution. (B) Partial alignment of Arabidopsis and human PEX1 and PEX6 and human p97 (a related ATPase) above protein schematic illustrations depicting the locations of pex1-1 and previously described pex6 missense alleles (35, 37, 44). The complete AAA domains are in yellow, with the Walker A (A), Walker B (B), and second region of homology (SRH) domains highlighted in navy blue. (C) pex1-1 is semidominant; the PEX1/pex1-1 heterozygote partially suppresses pex6-1 sucrose dependence and IBA resistance. Seedlings were grown on the indicated media for 1 d under yellow-filtered light before moving to darkness for 5 d. Bars indicate mean hypocotyl lengths, and error bars indicate SD (n ≥ 13 except for pex6-1 PEX1 and pex6-1 pex1-1 from the segregating parent, for which n ≥ 5). Means not sharing a letter above the bar are significantly different as determined by one-way ANOVA (P < 0.001).

Fig. 2.

Overexpressing PEX1 in pex1-1 pex6-1 phenocopies pex6-1 defects; pex1-1 does not restore pxa1-1 growth. (A) Expressing HA-PEX1 increases resistance to the inhibitory effects of IBA and hypocotyl elongation dependence on exogenous sucrose of dark-grown pex1-1 pex6-1 seedlings. Two independent pex1-1 pex6-1 35S:HA-PEX1 lines (A and B) are shown. Seedlings were grown as in the legend to Fig. 1C. Bars indicate mean hypocotyl lengths (n ≥ 13), and error bars indicate SD. (B) PTS2-processing defects of light-grown pex1-1 pex6-1 seedlings are worsened by HA-PEX1 expression. An immunoblot of 8-d-old light-grown seedling extracts was serially probed with antibodies to the indicated proteins. For thiolase and PMDH, precursor (p) and mature (m) proteins are indicated. (C) PEX1 and PEX6 levels resemble WT levels in pex1-1. An immunoblot of 8-d-old light-grown seedling extracts was serially probed with antibodies to the indicated proteins. For B and C, the positions of molecular mass markers (in kilodaltons) are indicated on the right and HSC70 is a loading control. (D) pex1-1 does not improve growth of pxa1-1 seedlings in the absence of sucrose. Seedlings were grown as in the legend to Fig. 1C. Bars indicate mean hypocotyl lengths (n ≥ 13), and error bars indicate SD. In A and D, means not sharing a letter above the bar are significantly different as determined by one-way ANOVA (P < 0.001).

Fig. 3.

pex1-1 ameliorates pex6-1 and pex6-3 and worsens pex26-1 defects without restoring PEX5 levels. (A) pex1-1 increases the sensitivity of dark-grown pex6-1 and pex6-3 seedlings to the inhibitory effects of IBA and reduces dependence on exogenous sucrose for hypocotyl elongation but increases pex26-1 sucrose dependence. Seedlings were grown as in the legend to Fig. 1C. Bars indicate mean hypocotyl lengths (n ≥ 13), and error bars indicate SD. (B) pex1-1 increases sensitivity of dark-grown pex6-1 and pex6-3 seedlings to the inhibitory effects of 2,4-DB on hypocotyl elongation. Seedlings were grown as in the legend to Fig. 1C. Bars indicate mean hypocotyl lengths (n ≥ 12), and error bars indicate SD. (C) pex1-1 increases sensitivity of light-grown pex6-3 seedlings to the stimulatory effects of IBA on lateral root production. Seedlings were grown on control medium for 4 d followed by 4 d on medium with or without 10 μM IBA under constant yellow-filtered light. Bars indicate mean lateral root densities (n ≥ 8), and error bars indicate SD. In A–C, means not sharing a letter above the bar are significantly different as determined by one-way ANOVA (P < 0.001). (D) pex1-1 differentially impacts PTS2 processing and peroxisomal protein levels in pex6 and pex26-1 seedlings. Immunoblots of 4- (Top), 5- (Middle), and 15-d-old (Bottom) light-grown seedling extracts were serially probed with antibodies to the indicated proteins. The positions of molecular mass markers (in kilodaltons) are indicated on the left. For thiolase and PMDH, precursor (p) and mature (m) proteins are indicated. HSC70 is a loading control. An asterisk indicates a cross-reacting band in the ICL panels.

Fig. 4.

pex1-1 lessens pex6 growth defects. (A and B) pex1-1 resembles WT and restores pex6 mutant defects in size and pigmentation. Seedlings at 7 d old (A) and 14 d old (B) were grown on sucrose-containing medium before photography (Scale bar: 1 cm.) (C) pex1-1 resembles WT and rescues pex6-1 and pex6-3 defects in adult size. Plants were transferred to soil after 14 d on sucrose-containing medium and photographed after an additional 4 wk in light. (Scale bar: 5 cm.)

Fig. 5.

pex1-1 slightly improves pex6-1 and pex6-3 GFP-PTS1 import and improves oil body utilization in pex6 mutants but does not improve oil body utilization in pex26-1. Confocal images of cotyledon epidermal cells (A) and hypocotyl cells (B) of 5-d-old seedlings carrying 35S:GFP-PTS1 (green) stained with Nile red (magenta). Emissions were collected at 490–519 nm for GFP and 587–643 nm for Nile red. Seedlings were screened for germination 2 d after plating, and individuals that had germinated by day 2 were imaged on day 5. (Scale bar: 20 μm.)

Fig. 6.

PEX5 remains excessively membrane-associated in pex1-1 pex6-1. Homogenates (H) prepared from 6-d-old dark-grown WT, pex1-1, pex6-1, and pex1-1 pex6-1 seedlings were separated by centrifugation to isolate cytosolic supernatant (S) and an organellar pellet, which was resuspended and recentrifuged to provide a final organellar pellet (P) fraction. Fractions were subjected to immunoblotting with the indicated antibodies. HSC70 is cytosolic, and mitochondrial (mito) ATP synthase subunit α and PEX14 localize in the organelle fraction. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

Fig. 7.

pex1-1 and overexpressing PEX5 additively restore pex6-1 defects. (A and B) Overexpressing PEX5 improves pex1-1 pex6-1 dark-grown (A) and light-grown (B) physiology. Plant lines with (+) and without (−) 35S:PEX5 were grown on the indicated media for 1 d under yellow-filtered light before moving to darkness for 5 d (A) or were grown for 8 d under continuous yellow-filtered light (B). Bars indicate mean hypocotyl lengths (A; n ≥ 14) or root lengths (B; n ≥ 13), and error bars indicate SD. Means not sharing a letter above the bar are significantly different as determined by one-way ANOVA (P < 0.001). (C) Overexpressing PEX5 improves PTS2 processing in pex1-1 pex6-1 seedlings. An immunoblot of 8-d-old light-grown seedling extracts was serially probed with antibodies to the indicated proteins. Precursor (p) and mature (m) PMDH are indicated. HSC70 is a loading control.

Fig. 8.

pex1-1 augments benefits provided to pex6-1 by preventing autophagy without increasing PEX5 levels. (A) pex1-1, atg7-3, and pex13-1 improve pex6-1 IBA responsiveness and growth without sucrose, and pex1-1 further increases atg7-3 benefits. Seedlings were grown as in the legend to Fig. 1C. Bars indicate mean hypocotyl lengths (n = 16), and error bars indicate SD. Means not sharing a letter above the bar are significantly different as determined by one-way ANOVA (P < 0.001). (B) Preventing autophagy increases PTS2 processing in pex6-1, and this increase is further improved by pex1-1. An immunoblot of 8-d-old light-grown seedling extracts was serially probed with antibodies to the indicated proteins. Precursor (p) and mature (m) PMDH are indicated. HSC70 is a loading control. (C) Preventing autophagy and pex1-1 synergistically improve GFP-PTS1 import in pex6-1. Confocal images of cotyledon epidermal and hypocotyl cells were collected after staining 5-d-old seedlings carrying 35S:GFP-PTS1 (green) with Nile red (magenta). Emissions were collected at 490–526 nm for GFP and 586–643 nm for Nile red.