Most eukaryotic cells require peroxisomes, organelles housing fatty acid b-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired b-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.

SUMMARY Catabolism of fatty acids stored in oil bodies is essential for Arabidopsis seed germination and seedling development. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail-anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling β-oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by proteasome inhibitor treatment or by combining pex26 with peroxisome-associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when PEX6 or PEX26 function is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex.

A variety of metabolic pathways are sequestered in peroxisomes, conserved organelles that are essential for human and plant survival. Peroxin (PEX) proteins generate and maintain peroxisomes. The PEX1 ATPase facilitates recycling of the peroxisome matrix protein receptor PEX5 and is the most commonly affected peroxin in human peroxisome biogenesis disorders. Here, we describe the isolation and characterization of, to our knowledge, the first Arabidopsis (Arabidopsis thaliana) pex1 missense alleles: pex1-2 and pex1-3. pex1-2 displayed peroxisome-related defects accompanied by reduced PEX1 and PEX6 levels. These pex1-2 defects were exacerbated by growth at high temperature and ameliorated by growth at low temperature or by PEX6 overexpression, suggesting that PEX1 enhances PEX6 stability and vice versa. pex1-3 conferred embryo lethality when homozygous, confirming that PEX1, like several other Arabidopsis peroxins, is essential for embryogenesis. pex1-3 displayed symptoms of peroxisome dysfunction when heterozygous; this semidominance is consistent with PEX1 forming a heterooligomer with PEX6 that is poisoned by pex1-3 subunits. Blocking autophagy partially rescued PEX1/pex1-3 defects, including the restoration of normal peroxisome size, suggesting that increasing peroxisome abundance can compensate for the deficiencies caused by pex1-3 and that the enlarged peroxisomes visible in PEX1/pex1-3 may represent autophagy intermediates. Overexpressing PEX1 in wild-type plants impaired growth, suggesting that excessive PEX1 can be detrimental. Our genetic, molecular, and physiological data support the heterohexamer model of PEX1-PEX6 function in plants.

Peroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import.

Plant peroxisomes house conserved functions such as β-oxidation and hydrogen peroxide decomposition along with specialized tasks including hormone metabolism and photorespiration. Phenotypes stemming from defects in these pathways have been exploited to isolate and characterize peroxisome-defective mutants in the reference plant Arabidopsis thaliana. Because peroxisome function is essential for plant viability, partial loss-of-function alleles have been frequently recovered from forward genetic screens. Analysis of these mutants has revealed the broad outlines of matrix protein import in plants and that these processes may be more similar between plants and mammals than between plants and yeast. Here we review matrix protein import into plant peroxisomes and the emerging understanding of how these matrix proteins may be degraded when they are damaged or no longer needed.