In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes

Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM> and SKV> (where > indicates the stop codon) as new PTS1s and the nonapeptide RVx₅HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.     

Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments

Peroxisomes are ubiquitous "multipurpose" organelles of eukaryotic cells. Their matrix enzymes catalyze mainly catabolic and anabolic reactions of lipid metabolism, thus contributing to the regulation of lipid homeostasis. Since most metabolites must be actively transported across the peroxisomal membrane and since individual proteins and protein complexes play functional roles in such transport processes, we analyzed the peroxisomal membrane proteome. Benzyldimethyl-n-hexadecylammoniumchloride (16-BAC)/SDS-2-D-PAGE and mass spectrometry were used to characterize the proteomes of highly purified "light" and "heavy" peroxisomes of rat liver obtained by density gradient centrifugation. In both populations, the major integral membrane proteins could be detected in high concentrations, verifying 16-BAC/SDS-2-D-PAGE as a suitable tool for the preparation of membrane proteomes destined for mass spectrometric analysis. Both reliable and reproducible detection of a distinct set of microsomal (ER) membrane proteins, including microsomal glutathione-S-transferase (mGST), in light and heavy peroxisomal fractions was also possible. Compared with the abundance of most microsomal membrane proteins, we found mGST to be specifically enriched in peroxisomal membrane fractions. Furthermore, C terminus epitope-tagged mGST versions were localized at least in part to peroxisomes in different mammalian cell lines. Taken together, these data suggest that the peroxisomal GST is not a mere ER-contaminant, but a bona fide protein comprising the membrane proteome of both intracellular compartments. In addition, we could detect several mitochondrial proteins in light peroxisome fractions. This finding may likely indicate a physical association of light peroxisomes with mitochondria, since the organelles could be partly separated by mechanical stress. Whether this association is of functional importance awaits further investigation.     

Proteome analysis of peroxisomes from dark-treated senescent Arabidopsis leaves

Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide,metabolism of fatty acids, photorespiration, and the biosynthesis of plant hormones. Plant peroxisomes have been traditionally classified into three major subtypes, and in-depth mass spectrometry(MS)-based proteomics has been performed to explore the proteome of the two major subtypes present in green leaves and etiolated seedlings. Here, we carried out a comprehensive proteome analysis of peroxisomes from Arabidopsis leaves given a 48-h dark treatment.Our goal was to determine the proteome of the third major subtype of plant peroxisomes from senescent leaves, and further catalog the plant peroxisomal proteome. We identifieda total of 111 peroxisomal proteins and verified the peroxisomal localization for six new proteins with potential roles in fatty acid metabolism and stress response by in vivo targeting analysis. Metabolic pathways compartmentalized in the three major subtypes of peroxisomes were also compared, which revealed a higher number of proteins involved in the detoxification of reactive oxygen species in peroxisomes from senescent leaves. Our study takes an important step towards mapping the ful function of plant peroxisomes.     

Toward a definition of the complete proteome of plant peroxisomes: Where experimental proteomics must be complemented by bioinformatics

In the past few years, proteome analysis of Arabidopsis peroxisomes has been established by the complementary efforts of four research groups and has emerged as the major unbiased approach to identify new peroxisomal proteins on a large scale. Collectively, more than 100 new candidate proteins from plant peroxisomes have been identified, including long-awaited low-abundance proteins. More than 50 proteins have been validated as peroxisome targeted, nearly doubling the number of established plant peroxisomal proteins. Sequence homologies of the new proteins predict unexpected enzyme activities, novel metabolic pathways and unknown non-metabolic peroxisome functions. Despite this remarkable success, proteome analyses of plant peroxisomes remain highly material intensive and require major preparative efforts. Characterization of the membrane proteome or post-translational protein modifications poses major technical challenges. New strategies, including quantitative mass spectrometry methods, need to be applied to allow further identifications of plant peroxisomal proteins, such as of stress-inducible proteins. In the long process of defining the complete proteome of plant peroxisomes, the prediction of peroxisome-targeted proteins from plant genome sequences emerges as an essential complementary approach to identify additional peroxisomal proteins that are, for instance, specific to peroxisome variants from minor tissues and organs or to abiotically stressed model and crop plants.     

Two small protein families, DYNAMIN-RELATED PROTEIN3 and FISSION1, are required for peroxisome fission in Arabidopsis

Peroxisomes are multi-functional organelles that differ in size and abundance depending on the species, cell type, developmental stage, and metabolic and environmental conditions. The PEROXIN11 protein family and the DYNAMIN-RELATED PROTEIN3A (DRP3A) protein have been shown previously to play key roles in peroxisome division in Arabidopsis. To establish a mechanistic model of peroxisome division in plants, we employed forward and reverse genetic approaches to identify more proteins involved in this process. In this study, we identified three new components of the Arabidopsis peroxisome division apparatus: DRP3B, a homolog of DRP3A, and FISSION1A and 1B (FIS1A and 1B), two homologs of the yeast and mammalian FIS1 proteins that mediate the fission of peroxisomes and mitochondria by tethering the DRP proteins to the membrane. DRP3B is partially targeted to peroxisomes and causes defects in peroxisome fission when the gene function is disrupted. drp3A drp3B double mutants display stronger deficiencies than each single mutant parent with respect to peroxisome abundance, seedling establishment and plant growth, suggesting partial functional redundancy between DRP3A and DRP3B. In addition, FIS1A and FIS1B are each dual-targeted to peroxisomes and mitochondria; their mutants show growth inhibition and contain peroxisomes and mitochondria with incomplete fission, enlarged size and reduced number. Our results demonstrate that both DRP3 and FIS1 protein families contribute to peroxisome fission in Arabidopsis, and support the view that DRP and FIS1 orthologs are common components of the peroxisomal and mitochondrial division machineries in diverse eukaryotic species.     

Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins.

Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.     

Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms

We have established a protocol for the isolation of highly purified peroxisomes from mature Arabidopsis thaliana leaves and analyzed the proteome by complementary gel-based and gel-free approaches. Seventy-eight nonredundant proteins were identified, of which 42 novel proteins had previously not been associated with plant peroxisomes. Seventeen novel proteins carried predicted peroxisomal targeting signals (PTS) type 1 or type 2; 11 proteins contained PTS-related peptides. Peroxisome targeting was supported for many novel proteins by in silico analyses and confirmed for 11 representative full-length fusion proteins by fluorescence microscopy. The targeting function of predicted and unpredicted signals was investigated and SSL>, SSI>, and ASL> were established as novel functional PTS1 peptides. In contrast with the generally accepted confinement of PTS2 peptides to the N-terminal domain, the bifunctional transthyretin-like protein was demonstrated to carry internally a functional PTS2. The novel enzymes include numerous enoyl-CoA hydratases, short-chain dehydrogenases, and several enzymes involved in NADP and glutathione metabolism. Seven proteins, including beta-glucosidases and myrosinases, support the currently emerging evidence for an important role of leaf peroxisomes in defense against pathogens and herbivores. The data provide new insights into the biology of plant peroxisomes and improve the prediction accuracy of peroxisome-targeted proteins from genome sequences.     

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.     

Pex19p binds Pex30p and Pex32p at regions required for their peroxisomal localization but separate from their peroxisomal targeting signals

The assembly of proteins in the peroxisomal membrane is a multistep process requiring their recognition in the cytosol, targeting to and insertion into the peroxisomal membrane, and stabilization within the lipid bilayer. The peroxin Pex19p has been proposed to be either the receptor that recognizes and targets newly synthesized peroxisomal membrane proteins (PMP) to the peroxisome or a chaperone required for stabilization of PMPs at the peroxisomal membrane. Differentiating between these two roles for Pex19p could be achieved by determining whether the peroxisomal targeting signal (PTS) and the region of Pex19p binding of a PMP are the same or different. We addressed the role for Pex19p in the assembly of two PMPs, Pex30p and Pex32p, of the yeast Saccharomyces cerevisiae. Pex30p and Pex32p control peroxisome size and number but are dispensable for peroxisome formation. Systematic truncations from the carboxyl terminus, together with in-frame deletions of specific regions, have identified PTSs essential for targeting Pex30p and Pex32p to peroxisomes. Both Pex30p and Pex32p interact with Pex19p in regions that do not overlap with their PTSs. However, Pex19p is required for localizing Pex30p and Pex32p to peroxisomes, because mutations that disrupt the interaction of Pex19p with Pex30p and Pex32p lead to their mislocalization to a compartment other than peroxisomes. Mutants of Pex30p and Pex32p that localize to peroxisomes but produce cells exhibiting the peroxisomal phenotypes of cells lacking these proteins demonstrate that the regions in these proteins that control peroxisomal targeting and cell biological activity are separable. Together, our data show that the interaction of Pex19p with Pex30p and Pex32p is required for their roles in peroxisome biogenesis and are consistent with a chaperone role for Pex19p in stabilizing or maintaining membrane proteins in peroxisomes.     

AraPerox. A database of putative Arabidopsis proteins from plant peroxisomes

To identify unknown proteins from plant peroxisomes, the Arabidopsis genome was screened for proteins with putative major or minor peroxisome targeting signals type 1 or 2 (PTS1 or PTS2), as defined previously (Reumann S [2004] Plant Physiol 135: 783-800). About 220 and 60 proteins were identified that carry a putative PTS1 or PTS2, respectively. To further support postulated targeting to peroxisomes, several prediction programs were applied and the putative targeting domains analyzed for properties conserved in peroxisomal proteins and for PTS conservation in homologous plant expressed sequence tags. The majority of proteins with a major PTS and medium to high overall probability of peroxisomal targeting represent novel nonhypothetical proteins and include several enzymes involved in beta-oxidation of unsaturated fatty acids and branched amino acids, and 2-hydroxy acid oxidases with a predicted function in fatty acid alpha-oxidation, as well as NADP-dependent dehydrogenases and reductases. In addition, large protein families with many putative peroxisomal isoforms were recognized, including acyl-activating enzymes, GDSL lipases, and small thioesterases. Several proteins are homologous to prokaryotic enzymes of a novel aerobic hybrid degradation pathway for aromatic compounds and proposed to be involved in peroxisomal biosynthesis of plant hormones like jasmonic acid, auxin, and salicylic acid. Putative regulatory proteins of plant peroxisomes include protein kinases, small heat shock proteins, and proteases. The information on subcellular targeting prediction, homology, and in silico expression analysis for these Arabidopsis proteins has been compiled in the public database AraPerox to accelerate discovery and experimental investigation of novel metabolic and regulatory pathways of plant peroxisomes.     

Peroxisome protein import: some answers, more questions

Recent advances in the study of plant peroxisomes are shedding new light on the importance of these organelles for plant development, and are revealing similarities and differences in peroxisome protein import pathways between plants, animals and fungi. For example, the import of matrix proteins that carry the PTS1 and PTS2 targeting signals is coupled in plants as it is in mammals, whereas these import pathways are separate in fungi. The expression of a human peroxisomal ATPase partially rescues the equivalent Arabidopsis mutant. Ubiquitination might play a role in receptor recycling in Saccharomyces cerevisiae and exciting progress is being made through studies of the targeting of membrane proteins.     

Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor

Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome- associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.     

The evolutionary origin of peroxisomes: an ER-peroxisome connection

The peroxisome is an essential eukaryotic organelle, crucial for lipid metabolism and free radical detoxification, development, differentiation, and morphogenesis from yeasts to humans. Loss of peroxisomes invariably leads to fatal peroxisome biogenesis disorders in man. The evolutionary origin of peroxisomes remains unsolved; proposals for either a symbiogenetic or cellular membrane invagination event are unconclusive. To address this question, we have probed with a peroxisomal proteome, an "ensemble" of 19 representative eukaryotic complete genomes. Molecular phylogenetic and sequence comparison tools allowed us to identify four proteins as peroxisomal markers for unequivocal in silico peroxisome detection. We have then detected the Apicomplexa phylum as the first group of organisms devoid of peroxisomes, in the presence of mitochondria. Finally, we deliver evidence against a prokaryotic ancestor of peroxisomes: (1) the peroxisomal membrane is composed of purely eukaryotic bricks and is thus useful to trace the eukaryotes in their evolutionary paths and (2) the peroxisomal matrix protein import system shares mechanistic similarities with the endoplasmic reticulum/proteasome degradation process, indicating a common evolutionary history.     

Identification of novel plant peroxisomal targeting signals by a combination of machine learning methods and in vivo subcellular targeting analyses

In the postgenomic era, accurate prediction tools are essential for identification of the proteomes of cell organelles. Prediction methods have been developed for peroxisome-targeted proteins in animals and fungi but are missing specifically for plants. For development of a predictor for plant proteins carrying peroxisome targeting signals type 1 (PTS1), we assembled more than 2500 homologous plant sequences, mainly from EST databases. We applied a discriminative machine learning approach to derive two different prediction methods, both of which showed high prediction accuracy and recognized specific targeting-enhancing patterns in the regions upstream of the PTS1 tripeptides. Upon application of these methods to the Arabidopsis thaliana genome, 392 gene models were predicted to be peroxisome targeted. These predictions were extensively tested in vivo, resulting in a high experimental verification rate of Arabidopsis proteins previously not known to be peroxisomal. The prediction methods were able to correctly infer novel PTS1 tripeptides, which even included novel residues. Twenty-three newly predicted PTS1 tripeptides were experimentally confirmed, and a high variability of the plant PTS1 motif was discovered. These prediction methods 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.     

The PEROXIN11 protein family controls peroxisome proliferation in Arabidopsis

PEROXIN11 (PEX11) is a peroxisomal membrane protein in fungi and mammals and was proposed to play a major role in peroxisome proliferation. To begin understanding how peroxisomes proliferate in plants and how changes in peroxisome abundance affect plant development, we characterized the extended Arabidopsis thaliana PEX11 protein family, consisting of the three phylogenetically distinct subfamilies PEX11a, PEX11b, and PEX11c to PEX11e. All five Arabidopsis PEX11 proteins target to peroxisomes, as demonstrated for endogenous and cyan fluorescent protein fusion proteins by fluorescence microscopy and immunobiochemical analysis using highly purified leaf peroxisomes. PEX11a and PEX11c to PEX11e behave as integral proteins of the peroxisome membrane. Overexpression of At PEX11 genes in Arabidopsis induced peroxisome proliferation, whereas reduction in gene expression decreased peroxisome abundance. PEX11c and PEX11e, but not PEX11a, PEX11b, and PEX11d, complemented to significant degrees the growth phenotype of the Saccharomyces cerevisiae pex11 null mutant on oleic acid. Heterologous expression of PEX11e in the yeast mutant increased the number and reduced the size of the peroxisomes. We conclude that all five Arabidopsis PEX11 proteins promote peroxisome proliferation and that individual family members play specific roles in distinct peroxisomal subtypes and environmental conditions and possibly in different steps of peroxisome proliferation.     

Structural,functional and genetic aspects of peroxisome biogenesis

Intracellular organelles, peroxisomes, occur in cells of most eukaryotic species. Human severe congenital disorders are associated with defective assembly and functioning of peroxisomes, which partly explains the attention of researchers paid to peroxisome biogenesis. It has been shown that peroxisomes are involved in the realization of eukaryotic developmental programs (in particular, neuroblast differentiation and postembryonic development). Cytobiochemical and electron-microscopic studies of peroxisomal mutations showed that the primary role in peroxisome biogenesis is played by synthesis of specific proteins (peroxins) and their transport and incorporation into peroxisome membranes. More than 30 peroxin-encoding genes have been examined. These proteins are synthesized on free polysomes and transported into peroxisomes by means of specific signaling peptides, PTS1, PTS2, and PTS3. The import of matrix proteins depends on at least two shuttle receptor proteins, Pex5p and Pex7p. Some proteins regulating peroxisome proliferation in cells have been identified.Translated from Genetika, Vol. 41, No. 2, 2005, pp. 149–165.Original Russian Text Copyright © 2005 by Kurbatova, Dutova, Trotsenko.     

Differential contribution of two peroxisomal protein receptors to the maintenance of peroxisomal functions in Arabidopsis

Peroxisomes in higher plant cells are known to differentiate in function depending on the cell type. Because of the functional differentiation, plant peroxisomes are subdivided into several classes, such as glyoxysomes and leaf peroxisomes. These peroxisomal functions are maintained by import of newly synthesized proteins containing one of two peroxisomal targeting signals known as PTS1 and PTS2. These targeting signals are known to be recognized by the cytosolic receptors, Pex5p and Pex7p, respectively. To demonstrate the contribution of Pex5p and Pex7p to the maintenance of peroxisomal functions in plants, double-stranded RNA constructs were introduced into the genome of Arabidopsis thaliana. Expression of the PEX5 and PEX7 genes was efficiently reduced by the double-stranded RNA-mediated interference in the transgenic Arabidopsis. The Pex5p-deficient Arabidopsis showed reduced activities for both glyoxysomal and leaf peroxisomal functions. An identical phenotype was observed in a transgenic Arabidopsis overexpressing functionally defective Pex5p. In contrast, the Pex7p-deficient Arabidopsis showed reduced activity for glyoxysomal function but not for leaf peroxisomal function. Analyses of peroxisomal protein import in the transgenic Arabidopsis revealed that Pex5p was involved in import of both PTS1-containing proteins and PTS2-containing proteins, whereas Pex7p contributed to the import of only PTS2-containing proteins. Overall, the results indicated that Pex5p and Pex7p play different roles in the maintenance of glyoxysomal and leaf peroxisomal functions in plants.     

Ubiquitination of the peroxisomal targeting signal type 1 receptor, Pex5p, suggests the presence of a quality control mechanism during peroxisomal matrix protein import

PEX genes encode proteins (peroxins) that are required for the biogenesis of peroxisomes. One of these peroxins, Pex5p, is the receptor for matrix proteins with a type 1 peroxisomal targeting signal (PTS1), which shuttles newly synthesized proteins from the cytosol into the peroxisome matrix. We observed that in various Saccharomyces cerevisiae pex mutants disturbed in the early stages of PTS1 import, the steady-state levels of Pex5p are enhanced relative to wild type controls. Furthermore, we identified ubiquitinated forms of Pex5p in deletion mutants of those PEX genes that have been implicated in recycling of Pex5p from the peroxisomal membrane into the cytosol. Pex5p ubiquitination required the presence of the ubiquitin-conjugating enzyme Ubc4p and the peroxins that are required during early stages of PTS1 protein import. Finally, we provide evidence that the proteasome is involved in the turnover of Pex5p in wild type yeast cells, a process that requires Ubc4p and occurs at the peroxisomal membrane. Our data suggest that during receptor recycling a portion of Pex5p becomes ubiquitinated and degraded by the proteasome. We propose that this process represents a conserved quality control mechanism in peroxisome biogenesis.     

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.