A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes

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.     

Recognition of a functional peroxisome type 1 target by the dynamic import receptor pex5p

Peroxisomes require the translocation of folded and functional target proteins of various sizes across the peroxisomal membrane. We have investigated the structure and function of the principal import receptor Pex5p, which recognizes targets bearing a C-terminal peroxisomal targeting signal type 1. Crystal structures of the receptor in the presence and absence of a peroxisomal target, sterol carrier protein 2, reveal major structural changes from an open, snail-like conformation into a closed, circular conformation. These changes are caused by a long loop C terminal to the 7-fold tetratricopeptide repeat segments. Mutations in residues of this loop lead to defects in peroxisomal import in human fibroblasts. The structure of the receptor/cargo complex demonstrates that the primary receptor-binding site of the cargo is structurally and topologically autonomous, enabling the cargo to retain its native structure and function.     

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.     

The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2-dependent protein transport to peroxisomes

Peroxisome biogenesis requires various complex processes including organelle division, enlargement and protein transport. We have been studying a number of Arabidopsis apm mutants that display aberrant peroxisome morphology. Two of these mutants, apm2 and apm4, showed green fluorescent protein fluorescence in the cytosol as well as in peroxisomes, indicating a decrease of efficiency of peroxisome targeting signal 1 (PTS1)-dependent protein transport to peroxisomes. Interestingly, both mutants were defective in PTS2-dependent protein transport. Plant growth was more inhibited in apm4 than apm2 mutants, apparently because protein transport was more severely decreased in apm4 than in apm2 mutants. APM2 and APM4 were found to encode proteins homologous to the peroxins PEX13 and PEX12, respectively, which are thought to be involved in transporting matrix proteins into peroxisomes in yeasts and mammals. We show that APM2/PEX13 and APM4/PEX12 are localized on peroxisomal membranes, and that APM2/PEX13 interacts with PEX7, a cytosolic PTS2 receptor. Additionally, a PTS1 receptor, PEX5, was found to stall on peroxisomal membranes in both mutants, suggesting that PEX12 and PEX13 are components that are involved in protein transport on peroxisomal membranes in higher plants. Proteins homologous to PEX12 and PEX13 have previously been found in Arabidopsis but it is not known whether they are involved in protein transport to peroxisomes. Our findings reveal that APM2/PEX13 and APM4/PEX12 are responsible for matrix protein import to peroxisomes in planta.     

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 Arabidopsis peroxisomal targeting signal type 2 receptor PEX7 is necessary for peroxisome function and dependent on PEX5

Plant peroxisomal proteins catalyze key metabolic reactions. Several peroxisome biogenesis PEROXIN (PEX) genes encode proteins acting in the import of targeted proteins necessary for these processes into the peroxisomal matrix. Most peroxisomal matrix proteins bear characterized Peroxisomal Targeting Signals (PTS1 or PTS2), which are bound by the receptors PEX5 or PEX7, respectively, for import into peroxisomes. Here we describe the isolation and characterization of an Arabidopsis peroxin mutant, pex7-1, which displays peroxisome-defective phenotypes including reduced PTS2 protein import. We also demonstrate that the pex5-1 PTS1 receptor mutant, which contains a lesion in a domain conserved among PEX7-binding proteins from various organisms, is defective not in PTS1 protein import, but rather in PTS2 protein import. Combining these mutations in a pex7-1 pex5-1 double mutant abolishes detectable PTS2 protein import and yields seedlings that are entirely sucrose-dependent for establishment, suggesting a severe block in peroxisomal fatty acid beta-oxidation. Adult pex7-1 pex5-1 plants have reduced stature and bear abnormally shaped seeds, few of which are viable. The pex7-1 pex5-1 seedlings that germinate have dramatically fewer lateral roots and often display fused cotyledons, phenotypes associated with reduced auxin response. Thus PTS2-directed peroxisomal import is necessary for normal embryonic development, seedling establishment, and vegetative growth.     

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.     

Disorders of peroxisome biogenesis due to mutations in PEX1: phenotypes and PEX1 protein levels

Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease (IRD) are clinically overlapping syndromes, collectively called "peroxisome biogenesis disorders" (PBDs), with clinical features being most severe in ZS and least pronounced in IRD. Inheritance of these disorders is autosomal recessive. The peroxisome biogenesis disorders are genetically heterogeneous, having at least 12 different complementation groups (CGs). The gene affected in CG1 is PEX1. Approximately 65% of the patients with PBD harbor mutations in PEX1. In the present study, we used SSCP analysis to evaluate a series of patients belonging to CG1 for mutations in PEX1 and studied phenotype-genotype correlations. A complete lack of PEX1 protein was found to be associated with severe ZS; however, residual amounts of PEX1 protein were found in patients with the milder phenotypes, NALD and IRD. The majority of these latter patients carried at least one copy of the common G843D allele. When patient fibroblasts harboring this allele were grown at 30 degrees C, a two- to threefold increase in PEX1 protein levels was observed, associated with a recovery of peroxisomal function. This suggests that the G843D missense mutation results in a misfolded protein, which is more stable at lower temperatures. We conclude that the search for the factors and/or mechanisms that determine the stability of mutant PEX1 protein by high-throughput procedures will be a first step in the development of therapeutic strategies for patients with mild PBDs.     

Biogenesis of nonspecific lipid transfer protein and sterol carrier protein x: studies using peroxisome assembly-defective pex cell mutants

Nonspecific lipid transfer protein (nsLTP; also called sterol carrier protein 2) with a molecular mass of 13 kDa is synthesized as a larger 15-kDa precursor (pre-nsLTP) with an N-terminal 20-amino acid extension presequence, as well as with the peroxisome targeting signal type 1 (PTS1), Ala-Lys-Leu, at the C terminus. The precursor pre-nsLTP is processed to mature nsLTP by proteolytic removal of the presequence, most likely after being imported into peroxisomes. Sterol carrier protein x (SCPx), a 59-kDa branched-chain fatty acid thiolase of peroxisomes, contains the entire pre-nsLTP moiety at the C-terminal part and is converted to the 46-kDa form and nsLTP after the transport to peroxisomes. We investigated which of these two potential topogenic sequences functions in biogenesis of nsLTP and SCPx. Morphological and biochemical analyses, making use of Chinese hamster ovary cell pex mutants such as the PTS1 receptor-impaired pex5 and PTS2 import-defective pex7, as well as green fluorescent protein chimeras, revealed that both pre-nsLTP and SCPx are imported into peroxisomes by the Pex5p-mediated PTS1 pathway. Nearly half of the pre-nsLTP remains in the cytosol, as assessed by subcellular fractionation of the wild-type Chinese hamster ovary cells. In an in vitro binding assay, only mature nsLTP, but not pre-nsLTP, from the cell lysates interacted with the Pex5p. It is likely, therefore, that modulation of the C-terminal PTS1 by the presequence gives rise to cytoplasmic localization of pre-nsLTP.     

Adenine phosphoribosyltransferase isoforms of Arabidopsis and their potential contributions to adenine and cytokinin metabolism

Adenine phosphoribosyltransferase (APT; EC 2.4.2.7) is a constitutively expressed enzyme involved in the one-step salvage of adenine to AMP. The Arabidopsis thaliana genome contains five sequences annotated as encoding APT or APT-like enzymes. Three of these have now been cloned, over-expressed and compared using kinetic analyses. At a cytosolic pH, all bind adenine efficiently based on their K_m values (0.8–2.6 µ M ), although APT1 metabolizes adenine at a rate 31–53 times faster than APT2 and APT3, respectively. Since APT also has a possible role in the interconversion of cytokinin bases to nucleotides, we characterized the activity of each isoform on zeatin, isopentenyladenine and benzyladenine. Based on their K_m values, APT2 and APT3 had much higher affinities than APT1 for all three cytokinins (15–440 µ M for APT2 and 3 vs. 1.8–2.5 m M for APT1); conversely the V_max values for APT2 and APT3 on these CK substrates showed the opposite trend, being 4- to 19-fold lower than those of APT1. Anti-peptide antibodies for APT1, APT2, and APT3 were prepared and used to examine the subcellular localization of each isoform. Based on these results, APT1 and APT3 appear to be cytosolic, while the localization of APT2 was inconclusive although sequence analysis implies that APT2 is also cytosolic. Each isoform was modelled against the crystal structure of APT from Leishmania donovani , and structural differences in substrate specificity-determining domains have been found. The estimated kinetic activities of these APTs suggest that they contribute primarily to adenine recycling, although an involvement in cytokinin interconversion cannot be discounted.     

Identification of six loci in which mutations partially restore peroxisome biogenesis and/or alleviate the metabolic defect of pex2 mutants in podospora

Peroxins (PEX) are proteins required for peroxisome biogenesis. Mutations in PEX genes cause lethal diseases in humans, metabolic defects in yeasts, and developmental disfunctions in plants and filamentous fungi. Here we describe the first large-scale screening for suppressors of a pex mutation. In Podospora anserina, pex2 mutants exhibit a metabolic defect [inability to grow on medium containing oleic acid (OA medium) as sole carbon source] and a developmental defect (inability to differentiate asci in homozygous crosses). Sixty-three mutations able to restore growth of pex2 mutants on OA medium have been analyzed. They fall in six loci (suo1 to suo6) and act as dominant, allele-nonspecific suppressors. Most suo mutations have pleiotropic effects in a pex2(+) background: formation of unripe ascospores (all loci except suo5 and suo6), impaired growth on OA medium (all loci except suo4 and suo6), or sexual defects (suo4). Using immunofluorescence and GFP staining, we show that peroxisome biogenesis is partially restored along with a low level of ascus differentiation in pex2 mutant strains carrying either the suo5 or the suo6 mutations. The data are discussed with respect to beta-oxidation of fatty acids, peroxisome biogenesis, and cell differentiation.     

Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants

Sulfur is an essential macro-element in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfur-related metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.     

Changes in carbohydrate metabolism and assimilate export in starch-excess mutants of Arabidopsis

The aim of this work was to investigate the effects on carbohydrate metabolism of a reduction in the capacity to degrade leaf starch in Arabidopsis. The major roles of leaf starch are to provide carbon for sucrose synthesis, respiration and, in developing leaves, for biosynthesis and growth. Wild-type plants were compared with plants of a starch-excess mutant line (sex4) deficient in a chloroplastic isoform of endoamylase. This mutant has a reduced capacity for starch degradation, leading to an imbalance between starch synthesis and degradation and the gradual accretion of starch as the leaves age. During the night the conversion of starch into sucrose in the mutant is impaired; the leaves of the mutant contained less sucrose than those of the wild type and there was less movement of ¹⁴C-label from starch to sucrose in radio-labelling experiments. Furthermore, the rate of assimilate export to the roots during the night was reduced in the mutant compared with the wild type. During the day however, photosynthetic partitioning was altered in the mutant, with less photosynthate partitioned into starch and more into sugars. Although the sucrose content of the leaves of the mutant was similar to the wild type during the day, the rate of export of sucrose to the roots was increased more than two-fold. The changes in carbohydrate metabolism in the mutant leaves during the day compensate partly for its reduced capacity to synthesize sucrose from starch during the night.     

The dynamin-like GTPase DLP1 is essential for peroxisome division and is recruited to peroxisomes in part by PEX11

Peroxisome division involves the conserved PEX11 peroxisomal membrane proteins and in yeast has been shown to require Vps1p, a dynamin-like protein. We show here that DLP1, the human homolog of the yeast DNM1 and VPS1 genes, plays an important role in peroxisome division in human cells. Disruption of DLP1 function by either RNA interference or overexpressing dominant negative DLP1 mutants causes a dramatic reduction in peroxisome abundance, although overexpression of functional DLP1 has no effect on peroxisome abundance. Overexpression of PEX11 induces peroxisome division in a multistep process involving elongation of preexisting peroxisomes followed by their division. We find that DLP1 is dispensable for the first phase of this process but essential for the second. Furthermore, we show that DLP1 associates with peroxisomes and that PEX11 overexpression recruits DLP1 to peroxisome membranes. However, we were unable to detect physical interaction between PEX11 and DLP1, and the stoichiometry of PEX11 and peroxisome-associated DLP1 was far less than 1:1. Based on these and other aspects, we propose that DLP1 performs an essential but transient role in peroxisome division and that PEX11 promotes peroxisome division by recruiting DLP1 to peroxisome membranes through an indirect mechanism.     

Functional conservation of plant secondary metabolic enzymes revealed by complementation of Arabidopsis flavonoid mutants with maize genes

Mutations in the transparent testa (tt) loci abolish pigment production in Arabidopsis seed coats. The TT4, TT5, and TT3 loci encode chalcone synthase, chalcone isomerase, and dihydroflavonol 4-reductase, respectively, which are essential for anthocyanin accumulation and may form a macromolecular complex. Here, we show that the products of the maize (Zea mays) C2, CHI1, and A1 genes complement Arabidopsis tt4, tt5, and tt3 mutants, restoring the ability of these mutants to accumulate pigments in seed coats and seedlings. Overexpression of the maize genes in wild-type Arabidopsis seedlings does not result in increased anthocyanin accumulation, suggesting that the steps catalyzed by these enzymes are not rate limiting in the conditions assayed. The expression of the maize A1 gene in the flavonoid 3' hydroxylase Arabidopsis tt7 mutant resulted in an increased accumulation of pelargonidin. We conclude that enzymes involved in secondary metabolism can be functionally exchangeable between plants separated by large evolutionary distances. This is in sharp contrast to the notion that the more relaxed selective constrains to which secondary metabolic pathways are subjected is responsible for the rapid divergence of the corresponding enzymes.     

Endoplasmic reticulum-directed Pex3p routes to peroxisomes and restores peroxisome formation in a Saccharomyces cerevisiae pex3Delta strain

Recent studies on the sorting of peroxisomal membrane proteins challenge the long-standing model in which peroxisomes are considered to be autonomous organelles that multiply by growth and division. Here, we present data lending support to the idea that the endoplasmic reticulum (ER) is involved in sorting of the peroxisomal membrane protein Pex3p, a protein required early in peroxisome biogenesis. First, we show that the introduction of an artificial glycosylation site into the N terminus of Pex3p leads to partial N-linked core glycosylation, indicative of insertion into the ER membrane. Second, when FLAG-tagged Pex3p is equipped with an ER targeting signal, it can restore peroxisome formation in pex3Delta cells. Importantly, FLAG antibodies that specifically recognize the processed Pex3p show that the signal peptide of the fusion protein is efficiently cleaved off and that the processed protein localizes to peroxisomes. In contrast, a Pex3p construct in which cleavage of the signal peptide is blocked by a mutation localizes to the ER and the cytosol and cannot complement pex3Delta cells. Together, these results strongly suggest that ER-targeted Pex3p indeed routes via the ER to peroxisomes, and we hypothesize that this pathway is also used by endogenous Pex3p.