Peroxisomal Targeting Signal Research Articles

The relevance of the non-canonical PTS1 of peroxisomal catalase

Catalase is sorted to peroxisomes via a C-terminal peroxisomal targeting signal 1 (PTS1), which binds to the receptor protein Pex5. Analysis of the C-terminal sequences of peroxisomal catalases from various species indicated that catalase never contains the typical C-terminal PTS1 tripeptide‐SKL, but invariably is sorted to peroxisomes via a non-canonical sorting sequence. We analyzed the relevance of the non-canonical PTS1 of catalase of the yeast Hansenula polymorpha (‐SKI). Using isothermal titration microcalorimetry, we show that the affinity of H. polymorpha Pex5 for a peptide containing ‐SKI at the C-terminus is 8-fold lower relative to a peptide that has a C-terminal ‐SKL. Fluorescence microscopy indicated that green fluorescent protein containing the ‐SKI tripeptide (GFP-SKI) has a prolonged residence time in the cytosol compared to GFP containing ‐SKL. Replacing the ‐SKI sequence of catalase into ‐SKL resulted in reduced levels of enzymatically active catalase in whole cell lysates together with the occurrence of catalase protein aggregates in the peroxisomal matrix. Moreover, the cultures showed a reduced growth yield in methanol-limited chemostats. Finally, we show that a mutant catalase variant that is unable to properly fold mislocalizes in protein aggregates in the cytosol. However, by replacing the PTS1 into ‐SKL the mutant variant accumulates in protein aggregates inside peroxisomes. Based on our findings we propose that the relatively weak PTS1 of catalase is important to allow proper folding of the enzyme prior to import into peroxisomes, thereby preventing the accumulation of catalase protein aggregates in the organelle matrix.

Molecular Requirements for Peroxisomal Targeting of Alanine-Glyoxylate Aminotransferase as an Essential Determinant in Primary Hyperoxaluria Type 1

Author SummaryPeroxisomes are cell organelles contain proteins involved in various aspects of metabolism. Peroxisome proteins translocate from their site of synthesis in the cytoplasm across the organelle membrane in a fully folded and functional form. One such protein is the enzyme alanine–glyoxylate aminotransferase (AGT). It contains a targeting signal in its C-terminus that is recognized by a receptor protein, Pex5p, in the cytoplasm, which allows its subsequent translocation into the peroxisome. Mutations in AGT cause a disease known as primary hyperoxaluria type 1, in which patients suffer irreversible kidney damage; this disease results, in many cases, from improper targeting of AGT into peroxisomes. To understand better the mechanism of AGT import into peroxisomes and the molecular basis of this disease, we have determined the crystal structure of the complex between AGT and its receptor Pex5p. The structure reveals how overlapping segments of the protein sequence are crucial for both receptor recognition and maintaining the folded structure of the enzyme. Subsequently, we created and studied several mutants of the enzyme, including mutants that are known to cause disease, and found that even minor folding defects in the enzyme prevent its recognition by Pexp5 and its import into peroxisomes. Our data thus provide novel insights into the consequences of mutations in AGT on the catalytic activity of the enzyme, as well as into the mechanisms that cause primary hyperoxaluria type 1.

Heterologous C-terminal signals effectively target fluorescent fusion proteins to leaf peroxisomes in diverse plant species

Peroxisomes are functionally diverse organelles that are wholly dependent on import of nuclear-encoded proteins. The signals that direct proteins into these organelles are either found at the C-terminus (type 1 peroxisomal targeting signal; PTS1) or N-terminus (type 2 peroxisomal targeting signal; PTS2) of the protein. Based on a limited number of tests in heterologous systems, PTS1 signals appear to be conserved across species. To further test the generality of this conclusion and to establish the extent to which the PTS1 signals can be relied on for biotechnological purposes across species, we tested two PTS1 signals for their ability to target fluorescent proteins in diverse plant species. Transient assays following microprojectile bombardment showed that the six amino acid PTS1 sequence (RAVARL) from spinach glycolate oxidase effectively targets green fluorescent fusion protein to the leaf peroxisomes in all 20 crops tested, including four monocots (sugarcane, wheat, corn and onion) and 16 dicots (carrot, cucumber, broccoli, tomato, lettuce, turnip, radish, cauliflower, cabbage, capsicum, celery, tobacco, petunia, beetroot, eggplant and coriander). Similarly, results indicated that the 10 amino acid PTS1 sequence (IHHPRELSRL) from pumpkin malate synthase effectively targets red fluorescent fusion protein to the leaf peroxisomes in all four crops tested including monocot (sugarcane) and dicot (cabbage, celery and pumpkin) species. These signal sequences should be useful metabolic engineering tools to direct recombinant proteins to the leaf peroxisomes in diverse plant species of biotechnological interest.

Glyoxylate Reductase Isoform 1 is Localized in the Cytosol and Not Peroxisomes in Plant Cells

Glyoxylate reductase (GLYR) is a key enzyme in plant metabolism which catalyzes the detoxification of both photorespiratory glyoxylate and succinic semialdehdye, an intermediate of the γ-aminobutyrate (GABA) pathway. Two isoforms of GLYR exist in plants, GLYR1 and GLYR2, and while GLYR2 is known to be localized in plastids, GLYR1 has been reported to be localized in either peroxisomes or the cytosol. Here, we reappraised the intracellular localization of GLYR1 in Arabidopsis thaliana L. Heynh (ecotype Lansberg erecta) using both transiently-transformed suspension cells and stably-transformed plants, in combination with fluorescence microscopy. The results indicate that GLYR1 is localized exclusively to the cytosol regardless of the species, tissue and/or cell type, or exposure of plants to environmental stresses that would increase flux through the GABA pathway. Moreover, the C-terminal tripeptide sequence of GLYR1, -SRE, despite its resemblance to a type 1 peroxisomal targeting signal, is not sufficient for targeting to peroxisomes. Collectively, these results define the cytosol as the intracellular location of GLYR1 and provide not only important insight to the metabolic roles of GLYR1 and the compartmentation of the GABA and photorespiratory pathways in plant cells, but also serve as a useful reference for future studies of proteins proposed to be localized to peroxisomes and/or the cytosol.

Role of peroxisomes in the biosynthesis and secretion of β-lactams and other secondary metabolites

Peroxisomes are eukaryotic organelles surrounded by a single bilayer membrane, containing a variety of proteins depending on the organism; they mainly perform degradation reactions of toxic metabolites (detoxification), catabolism of linear and branched-chain fatty acids, and removal of H(2)O(2) (formed in some oxidative processes) by catalase. Proteins named peroxins are involved in recruiting, transporting, and introducing the peroxisomal matrix proteins into the peroxisomes. The matrix proteins contain the peroxisomal targeting signals PTS1 and/or PTS2 that are recognized by the peroxins Pex5 and Pex7, respectively. Initial evidence indicated that the penicillin biosynthetic enzyme isopenicillinN acyltransferase (IAT) of Penicillium chrysogenum is located inside peroxisomes. There is now solid evidence (based on electron microscopy and/or biochemical data) confirming that IAT and the phenylacetic acid- and fatty acid-activating enzymes are also located in peroxisomes. Similarly, the Acremonium chrysogenum CefD1 and CefD2 proteins that perform the central reactions (activation and epimerization of isopenicillinN) of the cephalosporin pathway are targeted to peroxisomes. Growing evidence supports the conclusion that some enzymes involved in the biosynthesis of mycotoxins (e.g., AK-toxin), and the biosynthesis of signaling molecules in plants (e.g., jasmonic acid or auxins) occur in peroxisomes. The high concentration of substrates (in many cases toxic to the cytoplasm) and enzymes inside the peroxisomes allows efficient synthesis of metabolites with interesting biological or pharmacological activities. This compartmentalization poses additional challenges to the cell due to the need to import the substrates into the peroxisomes and to export the final products; the transporters involved in these processes are still very poorly known. This article focuses on new aspects of the metabolic processes occurring in peroxisomes, namely the degradation and detoxification processes that lead to the biosynthesis and secretion of secondary metabolites.

Association Between Peroxisomal Biogenesis Factor 7 and Autism Spectrum Disorders in a Korean Population

Autism spectrum disorder is a neurodevelopmental disorder characterized by deficits in social communication, impaired reciprocal social interaction, and repetitive patterns of behaviors or interests. Although the cause of autism spectrum disorder remains elusive, the present study identified peroxisomal biogenesis factor 7 (PEX7) as a gene associated with autism spectrum disorder, and this association was examined in a Korean population. PEX7 encodes a cytosolic receptor for peroxisome targeting signal 2 of peroxisomal matrix enzymes that are targeted to and translocated into the peroxisome. PEX7 defects are associated with rhizomelic chondrodysplasia punctata type 1 and Refsum disease. Mutations in PEX7 are related to a variety of mild to severe clinical symptoms, including mental retardation. The analysis of 9 intronic single nucleotide polymorphisms in 214 patients with autism spectrum disorder and 258 controls revealed the association of 2 single nucleotide polymorphisms and 1 haplotype with autism spectrum disorder (P < .05).

Identification and validation of Trypanosoma cruzi’s glycosomal adenylate kinase containing a peroxisomal targeting signal

Adenylate kinases are key enzymes involved in cell energy management. Trypanosomatid organisms have the largest number of isoforms found in a single cell, constituting a major difference with the mammalian hosts. In this work we study an adenylate kinase, TcADK3, the only Trypanosoma cruzi protein harboring the putative peroxisomal (glycosomal) targeting signal, “-CKL”. Parasites expressing GFP fused to TcADK3 showed a strong fluorescence in the glycosomes. The same result was obtained when the tripeptide “-CKL” was added at the C-terminus of the GFP, demonstrating that this signal is necessary and sufficient for targeting proteins to glycosomes. When this tripeptide was removed from the GFP-TcADK3 fusion protein, the fluorescence was re-localized in the cytoplasm. The CKL signal could be used for targeting foreign proteins to the glycosomes. This model also provides a useful tool to study glycosomes dynamics, morphology or number in living parasites in any stage of the life cycle.

Experimental and statistical post-validation of positive example EST sequences carrying peroxisome targeting signals type 1 (PTS1)

We recently developed the first algorithms specifically for plants to predict proteins carrying peroxisome targeting signals type 1 (PTS1) from genome sequences.1 As validated experimentally, the prediction methods are able to correctly predict unknown peroxisomal Arabidopsis proteins and to infer novel PTS1 tripeptides. The high prediction performance is primarily determined by the large number and sequence diversity of the underlying positive example sequences, which mainly derived from EST databases. However, a few constructs remained cytosolic in experimental validation studies, indicating sequencing errors in some ESTs. To identify erroneous sequences, we validated subcellular targeting of additional positive example sequences in the present study. Moreover, we analyzed the distribution of prediction scores separately for each orthologous group of PTS1 proteins, which generally resembled normal distributions with group-specific mean values. The cytosolic sequences commonly represented outliers of low prediction scores and were located at the very tail of a fitted normal distribution. Three statistical methods for identifying outliers were compared in terms of sensitivity and specificity.” Their combined application allows elimination of erroneous ESTs from positive example data sets. This new post-validation method will further improve the prediction accuracy of both PTS1 and PTS2 protein prediction models for plants, fungi, and mammals.

PredPlantPTS1: A Web Server for the Prediction of Plant Peroxisomal Proteins.

Prediction of subcellular protein localization is essential to correctly assign unknown proteins to cell organelle-specific protein networks and to ultimately determine protein function. For metazoa, several computational approaches have been developed in the past decade to predict peroxisomal proteins carrying the peroxisome targeting signal type 1 (PTS1). However, plant-specific PTS1 protein prediction methods have been lacking up to now, and pre-existing methods generally were incapable of correctly predicting low-abundance plant proteins possessing non-canonical PTS1 patterns. Recently, we presented a machine learning approach that is able to predict PTS1 proteins for higher plants (spermatophytes) with high accuracy and which can correctly identify unknown targeting patterns, i.e., novel PTS1 tripeptides and tripeptide residues. Here we describe the first plant-specific web server PredPlantPTS1 for the prediction of plant PTS1 proteins using the above-mentioned underlying models. The server allows the submission of protein sequences from diverse spermatophytes and also performs well for mosses and algae. The easy-to-use web interface provides detailed output in terms of (i) the peroxisomal targeting probability of the given sequence, (ii) information whether a particular non-canonical PTS1 tripeptide has already been experimentally verified, and (iii) the prediction scores for the single C-terminal 14 amino acid residues. The latter allows identification of predicted residues that inhibit peroxisome targeting and which can be optimized using site-directed mutagenesis to raise the peroxisome targeting efficiency. The prediction server will be instrumental in identifying low-abundance and stress-inducible peroxisomal proteins and defining the entire peroxisomal proteome of Arabidopsis and agronomically important crop plants. PredPlantPTS1 is freely accessible at ppp.gobics.de.

Visualization of microbodies in Chlamydomonas reinhardtii

In Chlorophycean algal cells, these organelles are generally called microbodies because they lack the enzymes found in the peroxisomes of higher plants. Microbodies in some algae contain fewer enzymes than the peroxisomes of higher plants, and some unicellular green algae in Chlorophyceae such as Chlamydomonas reinhardtii do not possess catalase, an enzyme commonly found in peroxisomes. Thus, whether microbodies in Chlorophycean algae are similar to the peroxisomes of higher plants, and whether they use a similar transport mechanism for the peroxisomal targeting signal (PTS), remain unclear. To determine whether the PTS is present in the microbodies of Chlorophycean algae, and to visualize the microbodies in Chlamydomonas cells, we examined the sub-cellular localization of green fluorescent proteins (GFP) fused to several PTS-like sequences. We detected GFP compartments that were spherical with a diameter of 0.3-1.0μm in transgenic Chlamydomonas. Comparative analysis of the character of GFP-compartments observed by fluorescence microscopy and that of microbodies by electron microscopy indicated that the compartments were one and the same. The result also showed that the microbodies in Chlorophycean cells have a similar transport mechanism to that of peroxisomes of higher plants.

Identification of a novel isoform of DHRS4 protein with a nuclear localization signal

The DHRS4 gene encodes an NADP(H)-dependent retinol dehydrogenase/reductase (NRDR) and plays an important role in regulating the synthesis of retinoic acid. In the present study, we identified a novel splice RNA variant, designated NRDRA2, of the human DHRS4 gene by RT-PCR, 3′ RACE, and 5′ RACE. NRDRA2 mRNA lacked exons 4 and 6, and had a shift in the reading frame when compared to DHRS4 mRNA, resulting in loss of the peroxisomal targeting signal of NRDR and gain of a nuclear localization signal in the predicted NRDRA2 protein. Endogenous NRDRA2 protein was identified in the human cervical carcinoma cell line HeLa by immunoprecipitation and mass spectrometric assay. A green fluorescent protein reporter assay showed that NRDRA2 protein mainly localized to the nuclei, confirming the sequence at its C-terminus as a legitimate nuclear localization signal sequence. This study identifies the alternative transcript variant NRDRA2 encoding a subcellular nuclear localized NRDRA2 protein.

The PEX7-Mediated Peroxisomal Import System Is Required for Fungal Development and Pathogenicity in Magnaporthe oryzae

In eukaryotes, microbodies called peroxisomes play important roles in cellular activities during the life cycle. Previous studies indicate that peroxisomal functions are important for plant infection in many phytopathogenic fungi, but detailed relationships between fungal pathogenicity and peroxisomal function still remain unclear. Here we report the importance of peroxisomal protein import through PTS2 (Peroxisomal Targeting Signal 2) in fungal development and pathogenicity of Magnaporthe oryzae. Using an Agrobacterium tumefaciens-mediated transformation library, a pathogenicity-defective mutant was isolated from M. oryzae and identified as a T-DNA insert in the PTS2 receptor gene, MoPEX7. Gene disruption of MoPEX7 abolished peroxisomal localization of a thiolase (MoTHL1) containing PTS2, supporting its role in the peroxisomal protein import machinery. ΔMopex7 showed significantly reduced mycelial growth on media containing short-chain fatty acids as a sole carbon source. ΔMopex7 produced fewer conidiophores and conidia, but conidial germination was normal. Conidia of ΔMopex7 were able to develop appressoria, but failed to cause disease in plant cells, except after wound inoculation. Appressoria formed by ΔMopex7 showed a defect in turgor generation due to a delay in lipid degradation and increased cell wall porosity during maturation. Taken together, our results suggest that the MoPEX7-mediated peroxisomal matrix protein import system is required for fungal development and pathogenicity M. oryzae.

Alternative splicing directs dual localization of Candida albicans 6-phosphogluconate dehydrogenase to cytosol and peroxisomes

The pentose phosphate pathway (PPP) is the main source of NADPH in the cell and therefore essential for the maintenance of the redox balance and anabolic reactions. NADPH is produced by the two dehydrogenases in the oxidative branch of the PPP: glucose-6-phosphate dehydrogenase (Zwf1) and 6-phosphogluconate dehydrogenase (Gnd1). We observed that in the commensal fungus Candida albicans these two enzymes contain putative peroxisomal targeting signals (PTSs): Zwf1 has a putative PTS1, while the annotated intron of GND1 encodes a PTS2. By subcellular fractionation and fluorescence microscopy, we show that both enzymes have a dual localization in which the majority is cytosolic, but a small fraction is peroxisome associated. Analysis of GND1 transcripts revealed that dual targeting of Gnd1 is directed by alternative splicing resulting in two Gnd1 isoforms, one without targeting signals localized to the cytosol and one with an N-terminal PTS2 targeted to peroxisomes. To our knowledge, Gnd1 is the first example of dual targeting of a protein by alternative splicing in C.albicans. In silico analysis suggests that PTS-mediated peroxisomal targeting of Zwf1 and Gnd1 is conserved across closely related Candida species. We discuss putative functions of the peroxisomal oxidative PPP in these organisms.

Structural Requirements for Interaction of Peroxisomal Targeting Signal 2 and Its Receptor PEX7

The import of a subset of peroxisomal matrix proteins is mediated by the peroxisomal targeting signal 2 (PTS2). The results of our sequence and physical property analysis of known PTS2 signals and of a mutational study of the least characterized amino acids of a canonical PTS2 motif indicate that PTS2 forms an amphipathic helix accumulating all conserved residues on one side. Three-dimensional structural modeling of the PTS2 receptor PEX7 reveals a groove with an evolutionarily conserved charge distribution complementary to PTS2 signals. Mammalian two-hybrid assays and cross-complementation of a mutation in PTS2 by a compensatory mutation in PEX7 confirm the interaction site. An unstructured linker region separates the PTS2 signal from the core protein. This additional information on PTS2 signals was used to generate a PTS2 prediction algorithm that enabled us to identify novel PTS2 signals within human proteins and to describe KChIP4 as a novel peroxisomal protein.

Two Proteases, Trypsin Domain-containing 1 (Tysnd1) and Peroxisomal Lon Protease (PsLon), Cooperatively Regulate Fatty Acid β-Oxidation in Peroxisomal Matrix

The molecular mechanisms underlying protein turnover and enzyme regulation in the peroxisomal matrix remain largely unknown. Trypsin domain-containing 1 (Tysnd1) and peroxisomal Lon protease (PsLon) are newly identified peroxisomal matrix proteins that harbor both a serine protease-like domain and a peroxisome-targeting signal 1 (PTS1) sequence. Tysnd1 processes several PTS1-containing proteins and cleaves N-terminal presequences from PTS2-containing protein precursors. Here we report that knockdown of Tysnd1, but not PsLon, resulted in accumulation of endogenous β-oxidation enzymes in their premature form. The protease activity of Tysnd1 was inactivated by intermolecular self-conversion of the 60-kDa form to 15- and 45-kDa chains, which were preferentially degraded by PsLon. Peroxisomal β-oxidation of a very long fatty acid was significantly decreased by knockdown of Tysnd1 and partially lowered by PsLon knockdown. Taken together, these data suggest that Tysnd1 is a key regulator of the peroxisomal β-oxidation pathway via proteolytic processing of β-oxidation enzymes. The proteolytic activity of oligomeric Tysnd1 is in turn controlled by self-cleavage of Tysnd1 and degradation of Tysnd1 cleavage products by PsLon.

Cysteine-dependent Ubiquitination of Pex18p Is Linked to Cargo Translocation across the Peroxisomal Membrane

The peroxisomal matrix protein import is facilitated by cycling receptor molecules that shuttle between the cytosol and the peroxisomal membrane. In the yeast Saccharomyces cerevisiae, the import of proteins harboring a peroxisomal targeting signal of type II (PTS2) is mediated by the receptor Pex7p and its co-receptor Pex18p. Here we demonstrate that Pex18p undergoes two kinds of ubiquitin modifications. One of these ubiquitination events depends on lysines 13 and 20 and forces rapid Pex18p turnover by proteasomal degradation. A cysteine residue near the extreme Pex18p amino-terminus is required for the second type of ubiquitination. It turned out that this cysteine residue at position 6 is essential for the function of Pex18p in peroxisomal protein import but does not contribute to receptor-cargo association and binding to the peroxisomal import apparatus. However, in contrast to the wild-type protein, cysteine 6-mutated Pex18p is arrested in a membrane-protected state, whereas Pex7p is accessible in a protease protection assay. This finding indicates that Pex18p export is linked to cargo translocation, which supports the idea of an export-driven import of proteins into peroxisomes.

Deg proteases and their role in protein quality control and processing in different subcellular compartments of the plant cell

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent serine endopeptidases found in almost every organism. Database searches revealed that 16 Deg paralogues are encoded by the genome of Arabidopsis thaliana, six of which were experimentally shown to be located in chloroplasts, one in peroxisomes, one in mitochondria and one in the nucleus. Two more Deg proteases are predicted to reside in chloroplasts, five in mitochondria (one of them with a dual chloroplastidial/mitochondrial localization) and the subcellular location of one protein is uncertain. This review summarizes the current knowledge on the role of Deg proteases in maintaining protein homeostasis and protein processing in various subcompartments of the plant cell. The chloroplast Deg proteases are the best examined so far, especially with respect to their role in the degradation of photodamaged photosynthetic proteins and in biogenesis of photosystem II (PSII). A combined action of thylakoid lumen and stroma Deg proteases in the primary cleavage of photodamaged D1 protein from PSII reaction centre is discussed on the basis of a recently resolved crystal structure of plant Deg1. The peroxisomal Deg protease is a processing enzyme responsible for the cleavage of N-terminal peroxisomal targeting signals (PTSs). A. thaliana mutants lacking this enzyme show reduced peroxisomal β-oxidation, indicating for the first time the impact of protein processing on peroxisomal functions in plants. Much less data is available for mitochondrial and nuclear Deg proteases. Based on the available expression data we hypothesize a role in general protein quality control and during acquired heat resistance.

AWP1/ZFAND6 Functions in Pex5 Export by Interacting with Cys‐Monoubiquitinated Pex5 and Pex6 AAA ATPase

During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.

Comparative profiling of the peroxisomal proteome of wildtype and Pex7 knockout mice by quantitative mass spectrometry

We present a label-free quantitative proteomic approach for the study of kidney peroxisomes of Pex7 knockout mice which is a bona-fide model for the human disease rhizomelic chondrodysplasia punctata (RCDP). RCDP is an autosomal recessive human disorder caused by mutations in the PEX7 gene encoding for Pex7, the cytosolic receptor protein that is essential for the import of proteins containing a functional peroxisomal targeting signal (PTS)-type 2. In this work, we quantitatively followed hundreds of proteins through high density gradient fractions of wildtype (WT) and Pex7 knockout (Pex7−/−) mice by high resolution mass spectrometry. A set of candidate proteins with altered abundance was defined via statistical and quantitative assessment of protein profiles obtained from WT and Pex7−/− mice. The results obtained demonstrate the feasibility of this approach to identify proteins specifically affected in abundance by the deletion of Pex7. All three known PTS2 proteins, including acetyl-Coenzyme A acyltransferase, alkylglycerone phosphate synthase and phytanoyl-CoA hydroxylase were determined to be virtually absent in these fractions whereas KIAA0564, a so far uncharacterized protein, was barely detectable in peroxisomal fractions of Pex7−/− mice. Furthermore, we report numerous PTS1 proteins with increased abundance levels in Pex7−/− mice that fulfill essential functions in the β-oxidation of very long-chain fatty acids or the biosynthesis of ether-phospholipids in peroxisomes.

A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.