Although participation of PEROXIN11 (PEX11), FISSION1 (FISl), and DYNAMIN-RELATED PROTEIN (DRP) has been well established during induced peroxisome proliferation in response to external stimuli, their roles in cell cycle-associated constitutive replication/duplication have not been fully explored. Herein, bimolecular fluorescence complementation experiments with Arabidopsis thaliana suspension cells revealed homooligomerization of all five PEX11 isoforms (PEX11a-e) and heterooligomerizations of all five PEX11 isoforms with FIS1b, but not FIS1a nor DRP3A. Intracellular protein targeting experiments demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e. Simultaneous silencing of PEX11c-e or individual silencing of DRP3A, but not FIS1a nor FIS1b, resulted in ;40% reductions in peroxisome number. During G2 in synchronized cell cultures, peroxisomes sequentially enlarged, elongated, and then doubled in number, which correlated with peaks in PEX11, FIS1, and DRP3A expression. Overall, these data support a model for the replication of preexisting peroxisomes wherein PEX11c, PEX11d, and PEX11e act cooperatively during G2 to promote peroxisome elongation and recruitment of FIS1b to the peroxisome membrane, where DRP3A stimulates fission of elongated peroxisomes into daughter peroxisomes, which are then distributed between daughter cells.

Peroxisomes are organelles that sequester certain metabolic pathways; many of these pathways generate H 2 O 2 , which can damage proteins. However, little is known about how damaged or obsolete peroxisomal proteins are degraded. We exploit developmentally timed peroxisomal content remodeling in Arabidopsis thaliana to elucidate peroxisome-associated protein degradation. Isocitrate lyase (ICL) is a peroxisomal glyoxylate cycle enzyme necessary for early seedling development. A few days after germination, photosynthesis begins and ICL is degraded. We previously found that ICL is stabilized when a peroxisome-associated ubiquitinconjugating enzyme and its membrane anchor are both mutated, suggesting that matrix proteins might exit the peroxisome for ubiquitin-dependent cytosolic degradation. To identify additional components needed for peroxisome-associated matrix protein degradation, we mutagenized a line expressing GFP-ICL, which is degraded similarly to endogenous ICL, and identified persistent GFP-ICL fluorescence (pfl) mutants. We found three pfl mutants that were defective in PEROXIN14 (PEX14/At5g62810), which encodes a peroxisomal membrane protein that assists in importing proteins into the peroxisome matrix, indicating that proteins must enter the peroxisome for efficient degradation. One pfl mutant was missing the peroxisomal 3-ketoacyl-CoA thiolase encoded by the PEROXISOME DEFECTIVE1 (PED1/At2g33150) gene, suggesting that peroxisomal metabolism influences the rate of matrix protein degradation. Finally, one pfl mutant that displayed normal matrix protein import carried a novel lesion in PEROXIN6 (PEX6/At1g03000), which encodes a peroxisome-tethered ATPase that is involved in recycling matrix protein receptors back to the cytosol. The isolation of pex6-2 as a pfl mutant supports the hypothesis that matrix proteins can exit the peroxisome for cytosolic degradation.

Pex11 homologs and dynamin-related proteins uniquely regulate peroxisome division (cell-cycle-dependent duplication) and proliferation (cell-cycle-independent multiplication). Arabidopsis plants possess five Pex11 homologs designated in this study as AtPex11a, -b, -c, -d and -e. Transcripts for four isoforms were found in Arabidopsis plant parts and in cells in suspension culture; by contrast, AtPex11a transcripts were found only in developing siliques. Within 2.5 hours after biolistic bombardments, myc-tagged or GFP-tagged AtPex11 a, -b, -c, -d and -e individually sorted from the cytosol directly to peroxisomes; none trafficked indirectly through the endoplasmic reticulum. Both termini of myc-tagged AtPex11 b, -c, -d and -e faced the cytosol, whereas the N- and C-termini of myc-AtPex11a faced the cytosol and matrix, respectively. In AtPex11a- or AtPex11e-transformed cells, peroxisomes doubled in number. Those peroxisomes bearing myc-AtPex11a, but not myc-AtPex11e, elongated prior to duplication. In cells transformed with AtPex11c or AtPex11d, peroxisomes elongated without subsequent fission. In AtPex11b-transformed cells, peroxisomes were aggregated and rounded. A C-terminal dilysine motif, present in AtPex11c, -d and -e, was not necessary for AtPex11d-induced peroxisome elongation. However, deletion of the motif from myc-AtPex11e led to peroxisome elongation and fission, indicating that the motif in this isoform promotes fission without elongation. In summary, all five overexpressed AtPex11 isoforms sort directly to peroxisomal membranes where they individually promote duplication (AtPex11a, -e), aggregation (AtPex11b), or elongation without fission (AtPex11c, -d).

Peroxisomes are ubiquitous eukaryotic organelles housing diverse enzymatic reactions, including several that produce toxic reactive oxygen species. Although understanding of the mechanisms whereby enzymes enter peroxisomes with the help of peroxin (PEX) proteins is increasing, mechanisms by which damaged or obsolete peroxisomal proteins are degraded are not understood. We have exploited unique aspects of plant development to characterize peroxisome-associated protein degradation (PexAD) in Arabidopsis. Oilseed seedlings undergo a developmentally regulated remodeling of peroxisomal matrix protein composition in which the glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MLS) are replaced by photorespiration enzymes. We found that mutations expected to increase or decrease peroxisomal H2O2 levels accelerated or delayed ICL and MLS disappearance, respectively, suggesting that oxidative damage promotes peroxisomal protein degradation. ICL, MLS, and the ␤-oxidation enzyme thiolase were stabilized in the pex4 -1 pex22-1 double mutant, which is defective in a peroxisome-associated ubiquitin-conjugating enzyme and its membrane tether. Moreover, the stabilized ICL, thiolase, and an ICL-GFP reporter remained peroxisome associated in pex4 -1 pex22-1. ICL also was stabilized and peroxisome associated in pex6 -1, a mutant defective in a peroxisome-tethered ATPase. ICL and thiolase were mislocalized to the cytosol but only ICL was stabilized in pex5-10, a mutant defective in a matrix protein import receptor, suggesting that peroxisome entry is necessary for degradation of certain matrix proteins. Together, our data reveal new roles for PEX4, PEX5, PEX6, and PEX22 in PexAD of damaged or obsolete matrix proteins in addition to their canonical roles in peroxisome biogenesis.Arabidopsis thaliana ͉ organelle remodeling ͉ peroxisome ͉ protein turnover P eroxisomes are ubiquitous eukaryotic organelles that characteristically possess H 2 O 2 -producing oxidases and H 2 O 2 -decomposing catalases. Specific reactions housed in peroxisomes vary by species, developmental stage, and cell type. For example, young seedling peroxisomes contain glyoxylate cycle enzymes, whereas mature leaf peroxisomes contain photorespiration enzymes (1). Peroxisomal proteins are nuclearly encoded and inserted posttranslationally into the peroxisome matrix or membrane with the assistance of peroxins (peroxisome biogenesis proteins) (2, 3). Although matrix protein import into peroxisomes is increasingly understood, mechanisms for recognizing and eliminating damaged or obsolete peroxisomal proteins remain largely obscure. Damage to peroxisomal proteins can occur through interactions with reactive oxygen species (ROS) produced by peroxisomal oxidative reactions. Most peroxisomal ROS are detoxified by catalase and the ascorbate-glutathione cycle (4); however, some ROS inevitably damage peroxisomal proteins (5). Mechanisms for detecting and eliminating damaged peroxisomal proteins have not been described; however, removal of excess or nonfunctio...

Proteins are targeted to the peroxisome matrix via processes that are mechanistically distinct from those used by other organelles. Protein entry into peroxisomes requires peroxin (PEX) proteins, including early-acting receptor (e.g. PEX5) and docking peroxins (e.g. PEX13 and PEX14) and late-acting PEX5-recycling peroxins (e.g. PEX4 and PEX6). We examined genetic interactions among Arabidopsis peroxin mutants and found that the weak pex13-1 allele had deleterious effects when combined with pex5-1 and pex14-2, which are defective in early-acting peroxins, as shown by reduced matrix protein import and enhanced physiological defects. In contrast, combining pex13-1 with pex4-1 or pex6-1, which are defective in late-acting peroxins, unexpectedly ameliorated mutant growth defects. Matrix protein import remained impaired in pex4-1 pex13-1 and pex6-1 pex13-1, suggesting that the partial suppression of pex4-1 and pex6-1 physiological defects by a weak pex13 allele may result from restoring the balance between import and export of PEX5 or other proteins that are retrotranslocated from the peroxisome with the assistance of PEX4 and PEX6. Our results suggest that symptoms caused by pex mutants defective in late-acting peroxins may result not only from defects in matrix protein import but also from inefficient removal of PEX5 from the peroxisomal membrane following cargo delivery.