An Arabidopsis dynamin-related protein, DRP3A, controls both peroxisomal and mitochondrial division

Peroxisomes undergo dramatic changes in size, shape, number, and position within the cell, but the division process of peroxisomes has not been characterized. We screened a number of Arabidopsis mutants with aberrant peroxisome morphology (apm mutants). In one of these mutants, apm1, the peroxisomes are long and reduced in number, apparently as a result of inhibition of division. We showed that APM1 encodes dynamin-related protein 3A (DRP3A), and that mutations in APM1/DRP3A also caused aberrant morphology of mitochondria. The transient expression analysis showed that DRP3A is associated with the cytosolic side of peroxisomes. These findings indicate that the same dynamin molecule is involved in peroxisomal and mitochondrial division in higher plants. We also report that the growth of Arabidopsis, which requires the cooperation of various organelles, including peroxisomes and mitochondria, is repressed in apm1, indicating that the changes of morphology of peroxisomes and mitochondria reduce the efficiency of metabolism in these organelles.     

pex5 Mutants that differentially disrupt PTS1 and PTS2 peroxisomal matrix protein import in Arabidopsis

PEX5 and PEX7 are receptors required for the import of peroxisome-bound proteins containing one of two peroxisomal targeting signals (PTS1 or PTS2). To better understand the role of PEX5 in plant peroxisomal import, we characterized the Arabidopsis (Arabidopsis thaliana) pex5-10 mutant, which has a T-DNA insertion in exon 5 of the PEX5 gene. Sequencing results revealed that exon 5, along with the T-DNA, is removed in this mutant, resulting in a truncated pex5 protein. The pex5-10 mutant has germination defects and is completely dependent on exogenous Suc for early seedling establishment, based on poor utilization of seed-storage fatty acids. This mutant also has delayed development and reduced fertility, although adult pex5-10 plants appear normal. Peroxisomal metabolism of indole-3-butyric acid, propionate, and isobutyrate also is disrupted. The pex5-10 mutant has reduced import of both PTS1 and PTS2 proteins, and enzymatic processes that occur in peroxisomes are disrupted. To specifically study the import and importance of PTS1 proteins, we made a truncated PEX5 construct lacking the PTS1-binding region (PEX5(454)). Transformation of this construct into pex5-10 resulted in the rescue of PTS2 import, thereby creating a line with PTS1-specific import defects. The pex5-10 (PEX5(454)) plants still had developmental defects, although restoring PTS2 import resulted in a less severe mutant phenotype. Comparison of pex5-10 and pex5-10 (PEX5(454)) phenotypes can separate the import mechanisms for enzymes acting in different peroxisomal processes, including indole-3-butyric acid/2,4-dichlorophenoxybutyric acid oxidation, isobutyrate and propionate metabolism, and photorespiration.     

Pex20p functions as the receptor for non‐PTS1/non‐PTS2 acyl‐CoA oxidase import into peroxisomes of the yeast Yarrowia lipolytica

Most soluble proteins targeted to the peroxisomal matrix contain a C‐terminal peroxisome targeting signal type 1 (PTS1) or an N‐terminal PTS2 that is recognized by the receptors Pex5p and Pex7p, respectively. These receptors cycle between the cytosol and peroxisome and back again for multiple rounds of cargo delivery to the peroxisome. A small number of peroxisomal matrix proteins, including all six isozymes of peroxisomal fatty acyl‐CoA oxidase (Aox) of the yeast Yarrowia lipolytica, contain neither a PTS1 nor a PTS2. Pex20p has been shown to function as a co‐receptor for Pex7p in the import of PTS2 cargo into peroxisomes. Here we show that cells of Y. lipolytica deleted for the PEX20 gene fail to import not only the PTS2‐containing protein 3‐ketoacyl‐CoA thiolase (Pot1p) but also the non‐PTS1/non‐PTS2 Aox isozymes. Pex20p binds directly to Aox isozymes Aox3p and Aox5p, which requires the C‐terminal Wxxx(F/Y) motif of Pex20p. A W411G mutation in the C‐terminal Wxxx(F/Y) motif causes Aox isozymes to be mislocalized to the cytosol. Pex20p interacts physically with members of the peroxisomal import docking complex, Pex13p and Pex14p. Our results are consistent with a role for Pex20p as the receptor for import of the non‐PTS1/non‐PTS2 Aox isozymes into peroxisomes.     

A novel pex2 mutant: catalase-deficient but temperature-sensitive PTS1 and PTS2 import

We searched for Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by using peroxisome targeting sequence (PTS) of Pex3p (amino acid residues 1-40)-fused enhanced green fluorescent protein (EGFP). From mutagenized wild-type CHO-K1 cells stably expressing rat Pex2p and Pex3p(1-40)-EGFP, cell colonies resistant to the 9-(1(')-pyrene)nonanol/ultraviolet treatment were examined for intracellular location of peroxisomal proteins, including EGFP chimera, catalase, and matrix proteins with PTS types 1 and 2. One clone, ZPEG309, showed a distinct phenotype: import defect of catalase, but normal transport of PTS1 and PTS2 proteins at 37 degrees C. PTS1 and PTS2 import was abrogated when ZPEG309 was cultured at 39 degrees C. Genetic defect of ZPEG309 was a nonsense point mutation in a codon for Arg50 in CHO PEX2 and a mutation resulting in a C-terminal truncation of the introduced rat Pex2p. Therefore, ZPEG309 is a novel pex2, catalase-deficient mutant with temperature-sensitive PTS1 and PTS2 import.     

Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: evidence that PTS1 protein import is mediated by a cycling receptor

PEX5 encodes the type-1 peroxisomal targeting signal (PTS1) receptor, one of at least 15 peroxins required for peroxisome biogenesis. Pex5p has a bimodal distribution within the cell, mostly cytosolic with a small amount bound to peroxisomes. This distribution indicates that Pex5p may function as a cycling receptor, a mode of action likely to require interaction with additional peroxins. Loss of peroxins required for protein translocation into the peroxisome (PEX2 or PEX12) resulted in accumulation of Pex5p at docking sites on the peroxisome surface. Pex5p also accumulated on peroxisomes in normal cells under conditions which inhibit protein translocation into peroxisomes (low temperature or ATP depletion), returned to the cytoplasm when translocation was restored, and reaccumulated on peroxisomes when translocation was again inhibited. Translocation inhibiting conditions did not result in Pex5p redistribution in cells that lack detectable peroxisomes. Thus, it appears that Pex5p can cycle repeatedly between the cytoplasm and peroxisome. Altered activity of the peroxin defective in CG7 cells leads to accumulation of Pex5p within the peroxisome, indicating that Pex5p may actually enter the peroxisome lumen at one point in its cycle. In addition, we found that the PTS1 receptor was extremely unstable in the peroxin-deficient CG1, CG4, and CG8 cells. Altered distribution or stability of the PTS1 receptor in all cells with a defect in PTS1 protein import implies that the genes mutated in these cell lines encode proteins with a direct role in peroxisomal protein import.     

Disparate peroxisome‐related defects in Arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization

Catabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. 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 treatment with a proteasome inhibitor or by combining pex26 with peroxisome‐associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 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.     

The plant PTS1 receptor: similarities and differences to its human and yeast counterparts

Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix. We have cloned and sequenced the watermelon ( Citrullus vulgaris ) cDNA homologue to the PTS1 receptor gene (PEX5). Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W-X₂-(E/S)-(Y/F/Q) in its N-terminal half. According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes. Transformation of the Hansenula polymorpha peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes. These structures are labeled in immunogold experiments using antibodies against the Hansenula polymorpha integral membrane protein Pex3p, confirming their peroxisomal nature. The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes. However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p.     

A comparative study of peroxisomal structures in Hansenula polymorpha pex mutants

In a recent study, we performed a systematic genome analysis for the conservation of genes involved in peroxisome biogenesis (PEX genes) in various fungi. We have now performed a systematic study of the morphology of peroxisome remnants ('ghosts') in Hansenula polymorpha pex mutants (pex1-pex20) and the level of peroxins and matrix proteins in these strains. To this end, all available H. polymorpha pex strains were grown under identical cultivation conditions in glucose-limited chemostat cultures and analyzed in detail. The H. polymorpha pex mutants could be categorized into four distinct groups, namely pex mutants containing: (1) virtually normal peroxisomal structures (pex7, pex17, pex20); (2) small peroxisomal membrane structures with a distinct lumen (pex2, pex4, pex5, pex10, pex12, pex14); (3) multilayered membrane structures lacking apparent matrix protein content (pex1, pex6, pex8, pex13); and (4) no peroxisomal structures (pex3, pex19).     

The peroxisome biogenesis factors pex4p, pex22p, pex1p, and pex6p act in the terminal steps of peroxisomal matrix protein import

Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.     

Cloning of two splice variants of the rice PTS1 receptor, OsPex5pL and OsPex5pS, and their functional characterization using pex5-deficient yeast and Arabidopsis

Using the rice PEX14 cDNA as a bait in a yeast two-hybrid assay, two splice variants of the type I peroxisomal targeting signal (PTS1) receptor, OsPex5pL and OsPex5pS, were cloned from a pathogen-treated rice leaf cDNA library. The proteins were produced from a single gene by alternative splicing, which generated a full-length variant, OsPEX5L, and a variant that lacked exon 7, OsPEX5S. OsPex5pL contained 11 copies of the pentapeptide motif WXXXF/Y in its N-terminus, and seven tetratricopeptide repeats in its C-terminus. Expression of OsPEX5L and OsPEX5S predominantly occurred in leaf tissues, and was induced by various stresses, such as exposure to the pathogen Magnaporthe grisea, and treatment with fungal elicitor, methyl viologen, NaCl or hydrogen peroxide. The Arabidopsis T-DNA insertional pex5 mutant, Atpex5, which does not germinate in the absence of sucrose and was resistant to indole-3-butyric acid (IBA), was perfectly rescued by over-expression of OsPex5pL, but not by OsPex5pS. Using transient expression of OsPex5pL and OsPex5pS in the Atpex5 mutant, we show that OsPex5pL translocates both PTS1- and PTS2-containing proteins into the peroxisome by interacting with OsPex7p, whereas OsPex5pS is involved only in PTS1-dependent import in Arabidopsis.     

Arabidopsis ABERRANT PEROXISOME MORPHOLOGY9 is a peroxin that recruits the PEX1-PEX6 complex to peroxisomes

Peroxisomes have pivotal roles in several metabolic processes, such as the detoxification of H₂O₂ and β-oxidation of fatty acids, and their functions are tightly regulated by multiple factors involved in peroxisome biogenesis, including protein transport. This study describes the isolation of an embryonic lethal Arabidopsis thaliana mutant, aberrant peroxisome morphology9 (apem9), which is compromised in protein transport into peroxisomes. The APEM9 gene was found to encode an unknown protein. Compared with apem9 having the nucleotide substitution, the knockdown mutants showed severe defects in peroxisomal functions and plant growth. We showed that expression of APEM9 altered PEROXIN6 (PEX6) subcellular localization from the cytosol to peroxisomes. In addition, we showed that PEX1 and PEX6 comprise a heterooligomer and that this complex was recruited to peroxisomal membranes via protein–protein interactions of APEM9 with PEX6. These findings show that APEM9 functions as an anchoring protein, similar to Pex26 in mammals and Pex15p in yeast. Interestingly, however, the identities of amino acids among these anchoring proteins are quite low. These results indicate that although the association of the PEX1-PEX6 complex with peroxisomal membranes is essential for peroxisomal functions, the protein that anchors this complex evolved uniquely in plants.     

Reducing PEX13 expression ameliorates physiological defects of late-acting peroxin mutants

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.     

Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor

Many peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p). Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus. However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system. In this work, we analyzed the PTS of pumpkin catalase (Cat1). A full or truncated pumpkin Cat1 cDNA fused at the 3' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv. Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation. The cellular localization of GFP was analyzed by fluorescence microscopy. The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes. Surprisingly, the C-terminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal. We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1. Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system.     

Rapid isolation and characterization of CHO mutants deficient in peroxisome biogenesis using the peroxisomal forms of fluorescent proteins

We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.     

PEX16 contributions to peroxisome import and metabolism revealed by viable Arabidopsis pex16 mutants

Peroxisomes rely on peroxins(PEX proteins) for biogenesis, importing membrane and matrix proteins, and fission. PEX_16, which is implicated in peroxisomal membrane protein targeting and forming nascent peroxisomes from the endoplasmic reticulum(ER), is unusual among peroxins because it is inserted co-translationally into the ER and localizes to both ER and peroxisomal membranes. PEX_16 mutations in humans, yeast, and plants confer some common peroxisomal defects; however, apparent functional differences have impeded the development of a unified model for PEX_16 action. The only reported pex_16 mutant in plants, the Arabidopsis shrunken seed1 mutant, is inviable,complicating analysis of PEX_16 function after embryogenesis. Here, we characterized two viable Arabidopsis pex_16 alleles that accumulate negligible PEX_16 protein levels. Both mutants displayed impaired peroxisome function-slowed consumption of stored oil bodies, decreased import of matrix proteins, and increased peroxisome size. Moreover,one pex_16 allele exhibited reduced growth that could be alleviated by an external fixed carbon source, decreased responsiveness to peroxisomally processed hormone precursors, and worsened or improved peroxisome function in combination with other pex mutants. Because the mutations impact different regions of the PEX_16 gene, these viable pex_16 alleles allow assessment of the importance of Arabidopsis PEX_16 and its functional domains.     

PEX5 binds the PTS1 independently of Hsp70 and the peroxin PEX12

Most peroxisomal enzymes are targeted to peroxisomes by virtue of a type-1 peroxisomal targeting signal (PTS1) at their extreme C terminus. PEX5 binds the PTS1 through its C-terminal 40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Here we examined the PTS1-binding activity of purified, recombinant, full-length PEX5 using a fluorescence anisotropy-based assay. Like its C-terminal fragment, full-length tetrameric PEX5 exhibits high intrinsic affinity for the PTS1, with a K(d) of 35 nm for the peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO(-). The specificity of this interaction was demonstrated by the fact that PEX5 had no detectable affinity for a peptide in which the Lys was replaced with Glu, a substitution that inactivates PTS1 signals in vivo. Hsp70 has been found to regulate the affinity of PEX5 for a PTS1-containing protein, but we found that the kinetics of PEX5-PTS1 binding was unaffected by Hsp70, Hsp70 plus ATP, or Hsp70 plus ADP. In addition, we found that another protein known to interact with the PTS1-binding domain of PEX5, the PEX12 zinc RING domain, also had no discernable effect on PEX5-PTS1 binding kinetics. Taken together, these results suggest that the initial step in peroxisomal protein import, the recognition of enzymes by PEX5, is a relatively simple process and that Hsp70 most probably stimulates this process by catalyzing the folding of newly synthesized peroxisomal enzymes and/or enhancing the accessibility of their PTS1.     

Pex5p imports folded tetrameric catalase by interaction with pex13p

Human catalase forms a 240-kDa tetrameric complex and degrades H(2) O(2) in peroxisomes. Human catalase is targeted to peroxisomes by the interaction of its peroxisomal targeting signal type 1 (PTS1)-like KANL sequence with the cytosolic PTS1 receptor Pex5p. We show herein that human catalase tetramers are formed in the cytoplasm and that the expression of a PTS signal on each of the four subunits is not necessary for peroxisomal transport. We previously demonstrated that a Pex5p mutant defective in binding to Pex13p, designated Pex5p(Mut234), imports typical PTS1-type proteins but not catalase. This impaired catalase import is not rescued by replacing its C-terminal KANL sequence with a typical PTS1 sequence, SKL, indicating that the failure of catalase import in Mut234-expressing cells is not due to its weak PTS1. In contrast, several enzymatically inactive and monomeric mutants of catalase are efficiently imported in Mut234-expressing cells. Moreover, trimeric chloramphenicol acetyltransferase (CAT) harboring SKL is not imported in Pex5p(Mut234)-expressing cells, but CAT-SKL trimers are transported to peroxisomes in the wild-type cells. These findings suggest that the Pex5p-Pex13p interaction likely plays a pivotal role in the peroxisomal import of folded and oligomeric proteins.     

Functional classification of Arabidopsis peroxisome biogenesis factors proposed from analyses of knockdown mutants

In higher plants, peroxisomes accomplish a variety of physiological functions such as lipid catabolism, photorespiration and hormone biosynthesis. Recently, many factors regulating peroxisomal biogenesis, so-called PEX genes, have been identified not only in plants but also in yeasts and mammals. In the Arabidopsis genome, the presence of at least 22 PEX genes has been proposed. Here, we clarify the physiological functions of 18 PEX genes for peroxisomal biogenesis by analyzing transgenic Arabidopsis plants that suppressed the PEX gene expression using RNA interference. The results indicated that the function of these PEX genes could be divided into two groups. One group involves PEX1, PEX2, PEX4, PEX6, PEX10, PEX12 and PEX13 together with previously characterized PEX5, PEX7 and PEX14. Defects in these genes caused loss of peroxisomal function due to misdistribution of peroxisomal matrix proteins in the cytosol. Of these, the pex10 mutant showed pleiotropic phenotypes that were not observed in any other pex mutants. In contrast, reduced peroxisomal function of the second group, including PEX3, PEX11, PEX16 and PEX19, was induced by morphological changes of the peroxisomes. Cells of the pex16 mutant in particular possessed reduced numbers of large peroxisome(s) that contained unknown vesicles. These results provide experimental evidence indicating that all of these PEX genes play pivotal roles in regulating peroxisomal biogenesis. We conclude that PEX genes belonging to the former group are involved in regulating peroxisomal protein import, whereas those of the latter group are important in maintaining the structure of peroxisome.     

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

Peroxisomes are a family of organelles which have many unusual features. They can arise de novo from the endoplasmic reticulum by a still poorly characterized process, yet possess a unique machinery for the import of their matrix proteins. As peroxisomes lack DNA, their function, which is highly variable and dependent on developmental and/or environmental conditions, is determined by the post‐translational import of specific metabolic enzymes in folded or oligomeric states. The two classes of matrix targeting signals for peroxisomal proteins [PTS1 (peroxisomal targeting signal 1) and PTS2] are recognized by cytosolic receptors [PEX5 (peroxin 5) and PEX7 respectively] which escort their cargo proteins to, or possibly across, the peroxisome membrane. Although the membrane translocation mechanism remains unclear, it appears to be driven by thermodynamically favourable binding interactions. Recycling of the receptors from the peroxisome membrane requires ATP hydrolysis for two linked processes: ubiquitination of PEX5 (and the PEX7 co‐receptors in yeast) and the function of two peroxisome‐associated AAA (ATPase associated with various cellular activities) ATPases, which play a role in recycling or turnover of the ubiquitinated receptors. This review summarizes and integrates recent findings on peroxisome matrix protein import from yeast, plant and mammalian model systems, and discusses some of the gaps in our understanding of this remarkable protein transport system.     

Matrix proteins are inefficiently imported into Arabidopsis peroxisomes lacking the receptor-docking peroxin PEX14

Mutations in peroxisome biogenesis proteins (peroxins) can lead to developmental deficiencies in various eukaryotes. PEX14 and PEX13 are peroxins involved in docking cargo-receptor complexes at the peroxisomal membrane, thus aiding in the transport of the cargo into the peroxisomal matrix. Genetic screens have revealed numerous Arabidopsis thaliana peroxins acting in peroxisomal matrix protein import; the viable alleles isolated through these screens are generally partial loss-of-function alleles, whereas null mutations that disrupt delivery of matrix proteins to peroxisomes can confer embryonic lethality. In this study, we used forward and reverse genetics in Arabidopsis to isolate four pex14 alleles. We found that all four alleles conferred reduced PEX14 mRNA levels and displayed physiological and molecular defects suggesting reduced but not abolished peroxisomal matrix protein import. The least severe pex14 allele, pex14-3, accumulated low levels of a C-terminally truncated PEX14 product that retained partial function. Surprisingly, even the severe pex14-2 allele, which lacked detectable PEX14 mRNA and PEX14 protein, was viable, fertile, and displayed residual peroxisome matrix protein import. As pex14 plants matured, import improved. Together, our data indicate that PEX14 facilitates, but is not essential for peroxisomal matrix protein import in plants.