The putative Saccharomyces cerevisiae hydrolase Ldh1p is localized to lipid droplets

Here, we report the identification of a novel hydrolase in Saccharomyces cerevisiae. Ldh1p (systematic name, Ybr204cp) comprises the typical GXSXG-type lipase motif of members of the α/β-hydrolase family and shares some features with the peroxisomal lipase Lpx1p. Both proteins carry a putative peroxisomal targeting signal type1 (PTS1) and can be aligned with two regions of homology. While Lpx1p is known as a peroxisomal enzyme, subcellular localization studies revealed that Ldh1p is predominantly localized to lipid droplets, the storage compartment of nonpolar lipids. Ldh1p is not required for the function and biogenesis of peroxisomes, and targeting of Ldh1p to lipid droplets occurs independently of the PTS1 receptor Pex5p.     

Role of Pex21p for Piggyback Import of Gpd1p and Pnc1p into Peroxisomes of Saccharomyces cerevisiae

Proteins designated for peroxisomal protein import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here, we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, whereas Pex18p is especially important for oleate-induced import of PTS2 proteins. The glycerol-producing PTS2 protein glycerol-3-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol, and in the nucleus under osmotic stress conditions. We show the following: (i) Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD⁺ salvage pathway, Pnc1p; (ii) Pnc1p, a nicotinamidase without functional PTS2, is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions.     

Peroxisomal catalase in the methylotrophic yeast Candida boidinii: transport efficiency and metabolic significance

In this study we cloned CTA1, the gene encoding peroxisomal catalase, from the methylotrophic yeast Candida boidinii and studied targeting of the gene product, Cta1p, into peroxisomes by using green fluorescent protein (GFP) fusion proteins. A strain from which CTA1 was deleted (cta1Delta strain) showed marked growth inhibition when it was grown on the peroxisome-inducing carbon sources methanol, oleate, and D-alanine, indicating that peroxisomal catalase plays an important nonspecific role in peroxisomal metabolism. Cta1p carries a peroxisomal targeting signal type 1 (PTS1) motif, -NKF, in its carboxyl terminus. Using GFP fusion proteins, we found that (i) Cta1p is transported to peroxisomes via its PTS1 motif, -NKF; (ii) peroxisomal localization is necessary for Cta1p to function physiologically; and (iii) Cta1p is bimodally distributed between the cytosol and peroxisomes in methanol-grown cells but is localized exclusively in peroxisomes in oleate- and D-alanine-grown cells. In contrast, the fusion protein GFP-AKL (GFP fused to another typical PTS1 sequence, -AKL), in the context of CbPmp20 and D-amino acid oxidase, was found to localize exclusively in peroxisomes. A yeast two-hybrid system analysis suggested that the low transport efficiency of the -NKF sequence is due to a level of interaction between the -NKF sequence and the PTS1 receptor that is lower than the level of interaction with the AKL sequence. Furthermore, GFP-Cta1pDeltankf coexpressed with Cta1p was successfully localized in peroxisomes, suggesting that the oligomer was formed prior to peroxisome import and that it is not necessary for all four subunits to possess a PTS motif. Since the main physiological function of catalase is degradation of H2O2, suboptimal efficiency of catalase import may confer an evolutionary advantage. We suggest that the PTS1 sequence, which is found in peroxisomal catalases, has evolved in such a way as to give a higher priority for peroxisomal transport to peroxisomal enzymes other than to catalases (e.g., oxidases), which require a higher level of peroxisomal transport efficiency.     

Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha Pex14 null mutant

Hansenula polymorpha Deltapex14 cells are affected in peroxisomal matrix protein import and lack normal peroxisomes. Instead, they contain peroxisomal membrane remnants, which harbor a very small amount of the major peroxisomal matrix enzymes alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS). The bulk of these proteins is, however, mislocated in the cytosol. Here, we show that in Deltapex14 cells overproduction of the PTS1 receptor, Pex5p, leads to enhanced import of the PTS1 proteins AO and DHAS but not of the PTS2 protein amine oxidase. The import of the PTS1 protein catalase (CAT) was not stimulated by Pex5p overproduction. The difference in import behavior of AO and CAT was not related to their PTS1, since green fluorescent protein fused to the PTS1 of either AO or CAT were both not imported in Deltapex14 cells overproducing Pex5p. When produced in a wild type control strain, both proteins were normally imported into peroxisomes. In Deltapex14 cells overproducing Pex5p, Pex5p had a dual location and was localized in the cytosol and bound to the outer surface of the peroxisomal membrane. Our results indicate that binding of Pex5p to the peroxisomal membrane and import of certain PTS1 proteins can proceed in the absence of Pex14p.     

Involvement of Pex13p in Pex14p localization and peroxisomal targeting signal 2-dependent protein import into peroxisomes

Pex13p is the putative docking protein for peroxisomal targeting signal 1 (PTS1)-dependent protein import into peroxisomes. Pex14p interacts with both the PTS1- and PTS2-receptor and may represent the point of convergence of the PTS1- and PTS2-dependent protein import pathways. We report the involvement of Pex13p in peroxisomal import of PTS2-containing proteins. Like Pex14p, Pex13p not only interacts with the PTS1-receptor Pex5p, but also with the PTS2-receptor Pex7p; however, this association may be direct or indirect. In support of distinct peroxisomal binding sites for Pex7p, the Pex7p/Pex13p and Pex7p/ Pex14p complexes can form independently. Genetic evidence for the interaction of Pex7p and Pex13p is provided by the observation that overexpression of Pex13p suppresses a loss of function mutant of Pex7p. Accordingly, we conclude that Pex7p and Pex13p functionally interact during PTS2-dependent protein import into peroxisomes. NH2-terminal regions of Pex13p are required for its interaction with the PTS2-receptor while the COOH-terminal SH3 domain alone is sufficient to mediate its interaction with the PTS1-receptor. Reinvestigation of the topology revealed both termini of Pex13p to be oriented towards the cytosol. We also found Pex13p to be required for peroxisomal association of Pex14p, yet the SH3 domain of Pex13p may not provide the only binding site for Pex14p at the peroxisomal membrane.     

Peroxisome senescence in human fibroblasts

The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging.     

Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana

Leaf peroxisomes are present in greening cotyledons and contain enzymes of the glycolate pathway that functions in photorespiration. However, only a few leaf peroxisomal proteins, that is hydroxypyruvate reductase (HPR), glycolate oxidase (GO) and alanine:glyoxylate aminotransferase 1 (AGT1), have been characterized, and other functions in leaf peroxisomes have not been solved. To better understand the functions of leaf peroxisomes, we established a method to isolate leaf peroxisomes of greening cotyledons. We analyzed 53 proteins by MALDI-TOF MS and then identified 29 proteins. Among them, five proteins are related to the glycolate pathway, four proteins function in scavenging of hydrogen peroxide and additionally 20 novel leaf peroxisomal proteins were identified. In particular, protein kinases and protein phosphatase were first identified as peroxisomal proteins suggesting that protein phosphorylation is one of the regulatory mechanisms in leaf peroxisomes. Novel leaf peroxisomal proteins contained five PTS1-like proteins that have sequences where one amino acid is substituted with another one in PTS1 sequences. The PTS1 motif was suggested to have novel PTS1 sequences.     

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.     

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.     

Quantitative analysis of peroxisomal protein import in vitro

Protein import into the peroxisome matrix is mediated by peroxisome-targeting signals (PTSs). We have developed a novel, quantitative, in vitro assay for measuring peroxisomal import of PTS1-containing proteins. This enzyme-linked immunosorbent assay-based system utilizes semi-intact human A431 cells or fibroblasts and a biotinylated version of the PTS1-containing import substrate, luciferase. We show that biotinylated luciferase accumulated in peroxisomes in a time- and temperature-dependent fashion, in a reaction stimulated by exogenously added ATP, cytosol, and zinc. No import was detected in fibroblasts from a human patient belonging to complementation group 2, who suffered from the fatal peroxisomal disorder Zellweger syndrome and lacked a functional PTS1 receptor, Pex5p. Also, the reaction was significantly inhibited by antibodies to the zinc-finger protein, Pex2p. Several lines of evidence demonstrate that biotinylated luciferase was imported into the lumen of bona fide peroxisomes. (a) Biochemical fractionation of cells after the import reaction showed a time-dependent accumulation of the import substrate within intracellular organelles. (b) Confocal fluorescence microscopy indicated that imported biotinylated luciferase colocalized with the peroxisomal protein PMP70. (c) Visualization of the imported biotinylated luciferase by indirect fluorescence or indirect immunofluorescence required disruption of the peroxisomal membrane, indicating true import rather than binding to the outside of the organelle.     

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.     

The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide peroxisomal targeting signal.

The peroxisomal targeting signal 1 (PTS1), consisting of a C-terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms. Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery. We now report several new properties of this molecule which, taken together, show that it is the peroxisomal PTS1 receptor. (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the peroxisomal membrane, (ii) peroxisomes of wild-type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1-containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross-linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant). In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.     

The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTS1 import machinery.

We have cloned the Hansenula polymorpha PEX4 gene by functional complementation of a peroxisome-deficient mutant. The PEX4 translation product, Pex4p, is a member of the ubiquitin-conjugating enzyme family. In H.polymorpha, Pex4p is a constitutive, low abundance protein. Both the original mutant and the pex4 deletion strain (Deltapex4) showed a specific defect in import of peroxisomal matrix proteins containing a C-terminal targeting signal (PTS1) and of malate synthase, whose targeting signal is not yet known. Import of the PTS2 protein amine oxidase and the insertion of the peroxisomal membrane proteins Pex3p and Pex14p was not disturbed in Deltapex4 cells. The PTS1 protein import defect in Deltapex4 cells could be suppressed by overproduction of the PTS1 receptor, Pex5p, in a dose-response related manner. In such cells, Pex5p is localized in the cytosol and in peroxisomes. The peroxisome-bound Pex5p specifically accumulated at the inner surface of the peroxisomal membrane and thus differed from Pex5p in wild-type peroxisomes, which is localized throughout the matrix. We hypothesize that in H. polymorpha Pex4p plays an essential role for normal functioning of Pex5p, possibly in mediating recycling of Pex5p from the peroxisome to the cytosol.     

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.     

PTS1-independent sorting of peroxisomal matrix proteins by Pex5p

Most peroxisomal matrix proteins contain a peroxisomal targeting signal 1 (PTS1) for sorting to the correct organelle. This signal is located at the extreme C-terminus and generally consists of only three amino acids. The PTS1 is recognized by the receptor protein Pex5p. Several examples have been reported of peroxisomal matrix proteins that are sorted to peroxisomes via Pex5p, but lack a typical PTS1 tripeptide. In this contribution we present an overview of these so-called non-PTS1 proteins and discuss the current knowledge of the molecular mechanisms involved in their sorting.     

The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins

We identified a Saccharomyces cerevisiae peroxisomal membrane protein, Pex13p, that is essential for protein import. A point mutation in the COOH-terminal Src homology 3 (SH3) domain of Pex13p inactivated the protein but did not affect its membrane targeting. A two-hybrid screen with the SH3 domain of Pex13p identified Pex5p, a receptor for proteins with a type I peroxisomal targeting signal (PTS1), as its ligand. Pex13p SH3 interacted specifically with Pex5p in vitro. We determined, furthermore, that Pex5p was mainly present in the cytosol and only a small fraction was associated with peroxisomes. We therefore propose that Pex13p is a component of the peroxisomal protein import machinery onto which the mobile Pex5p receptor docks for the delivery of the selected PTS1 protein.     

The unusual extended C-terminal helix of the peroxisomal α/β-hydrolase Lpx1 is involved in dimer contacts but dispensable for dimerization

The yeast peroxisomal hydrolase Lpx1 belongs to the α/β-hydrolase superfamily. In the absence of Lpx1, yeast peroxisomes show an aberrant vacuolated morphology similar to what is found in peroxisomal disorder patients. Here, we present the crystal structure of Lpx1 determined at a resolution of 1.9 ?. The structure reveals the complete catalytic triad with an unusual location of the acid residue after strand β6 of the canonical α/β-hydrolase fold. A four-helix cap domain covers the active site. The interface between the α/β-hydrolase core and the cap domain forms the potential substrate binding site, which may also comprise the tunnel that leads into the protein interior and widens into a cavity. Two further tunnels connect the active site to the protein surface, potentially facilitating substrate access. Lpx1 is a homodimer. The α/β-hydrolase core folds of the two protomers form the dimer contact site. Further dimerization contacts arise from the mutual embracement of the cap domain of one protomer by the non-canonical C-terminal helix of the other, resulting in a total buried surface area of some 6000 ?2. The unusual C-terminal helix sticks out from the core fold to which it is connected by an extended flexible loop. We analyzed whether this helix is required for dimerization and for import of the dimer into peroxisomes using biochemical assays in vitro and a microscopy-based interaction assay in mammalian cells. Surprisingly, the C-terminal helix is dispensable for dimerization and dimer import. The unusually robust self-interaction suggests that Lpx1 is imported into peroxisomes as dimer.     

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.     

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.     

Identification of a cryptic N-terminal signal in Saccharomyces cerevisiae peroxisomal citrate synthase that functions in both peroxisomal and mitochondrial targeting

Saccharomyces cerevisiae has three distinct citrate synthases, two located in mitochondria (mature Cit1p and Cit3p) and one in peroxisomes (mature Cit2p). While the precursor of the major mitochondrial enzyme, Cit1p, has a signal for mitochondrial targeting at its N-terminus (MTS), Cit2p has one for peroxisomal targeting (PTS1) at its C-terminus. We have previously shown that the N-terminal segment of Cit2p is removed during import into peroxisomes [Lee, H.S. et al. (1994) Kor. J. Microbiol. 32, 558-564], which implied the presence of an additional N-terminal sorting signal. To analyze the function of the N-terminal region of Cit2p in protein trafficking, we constructed the N-terminal domain-swapped versions of Cit1p and Cit2p. Both fusions, Cit1::Cit2 and Cit2::Cit1, complemented the glutamate auxotrophy caused by the double-disruption of the CIT1 and CIT2 genes. In addition, part of the Cit2::Cit1 fusion protein, as well as Cit1::Cit2, was shown to be transported into both mitochondria and peroxisomes. The subcellular localization of the recombinant fusion proteins containing various N-terminal segments of Cit2p fused to a mutant version of green fluorescent protein (GFP2) was also examined. As a result, we found that the 20-amino acid N-terminal segment of Cit2p contains a cryptic cleavable targeting signal for both peroxisomes and mitochondria. In addition, we show that the peroxisomal import process mediated by the N-terminal segment of Cit2p was not affected by the disruption of either PEX5 (encoding PTS1 receptor) or PEX7 (encoding PTS2 receptor).