PTS1-independent targeting of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p

The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Δ3 and Δ19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.     

Peroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris

Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine _COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX–HA). Coexpression of AOX–HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX–HA present in octameric active AOX, suggesting that newly synthesized AOX–HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.     

Plant Peroxisomes: Molecular Basis of the Regulation of their Functions

β -oxidation, the glyoxylate cycle and the glycolate pathway for photorespiration. Recent molecular biological studies have revealed that most of these enzymes possess either one of two peroxisomal targeting signals (PTS) within their amino acid sequence. One of the signals, PTS1, is found at the carboxy-terminus, while the other, PTS2, is found within the amino-terminal presequence. Subsequent to the synthesis and folding of these enzymes in the cytosol, the targeting signal in the folded proteins may bind to the corresponding receptors. At present, only a receptor that recognizes PTS1 has been identified in higher plants. After the binding of the protein and the receptor, the protein complex may be recognized by docking proteins that exist in the peroxisomal membrane. The mechanisms responsible for the recognition of peroxisomal proteins are now under investigation. Genetic analyses of Arabidopsis mutants with defective peroxisomes may give us some clues to understanding the mechanisms of peroxisomal protein import. Received 18 November 1999/ Accepted in revised form 13 January 2000     

Proteome of plant peroxisomes: new perspectives on the role of these organelles in cell biology

Peroxisomes are cell organelles bounded by a single membrane with a basically oxidative metabolism. Peroxisomes house catalase and H₂O₂‐producing flavin‐oxidases as the main protein constituents. However, since their discovery in early fifties, a number of new enzymes and metabolic pathways have been reported to be also confined to these organelles. Thus, the presence of exo‐ and endo‐peptidases, superoxide dismutases, the enzymes of the plant ascorbate‐glutathione cycle plus ascorbate and glutathione, several NADP‐dehydrogenases, and also L‐arginine‐dependent nitric oxide synthase activity has evidenced the relevant role of these organelles in cell physiology. In recent years, the study of new functions of peroxisomes has become a field of intensive research in cell biology, and these organelles have been proposed to be a source of important signal molecules for different transduction pathways. In plants, peroxisomes participate in seed germination, leaf senescence, fruit maturation, response to abiotic and biotic stress, photomorphogenesis, biosynthesis of the plant hormones jasmonic acid and auxin, and in cell signaling by reactive oxygen and nitrogen species (ROS and RNS, respectively). In order to decipher the nature and specific role of the peroxisomal proteins in these processes, several approaches including in vivo and in vitro import assays and generation of mutants have been used. In the last decade, the development of genomics and the report of the first plant genomes provided plant biologists a powerful tool to assign to peroxisomes those proteins which harbored any of the two peroxisomal targeting signals (PTS, either PTS1 or PTS2) described so far. Unfortunately, those molecular approaches could not give any response to those proteins previously localized in plant peroxisomes by classical biochemical and cell biology methods that did not contain any PTS. However, more recently, proteomic studies of highly purified organelles have provided evidence of the presence in peroxisomes of new proteins not previously reported. Thus, the contribution of proteomic approaches to the biology of peroxisomes is essential, not only for elucidation of the mechanisms involved in the import of the PTS1‐ and PTS2‐independent proteins, but also to the understanding of the role of these organelles in the cell physiology of plant growth and development.     

A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase.

Several peroxisomal proteins do not contain the previously identified tripeptide peroxisomal targeting signal (PTS) at their carboxy-termini. One such protein is the peroxisomal 3-ketoacyl CoA thiolase, of which two types exist in rat [Hijikata et al. (1990) J. Biol. Chem., 265, 4600-4606]. Both rat peroxisomal thiolases are synthesized as larger precursors with an amino-terminal prepiece of either 36 (type A) or 26 (type B) amino acids, that is cleaved upon translocation of the enzyme into the peroxisome. The prepieces are necessary for import of the thiolases into peroxisomes because expression of an altered cDNA encoding only the mature thiolase, which lacks any prepiece, results in synthesis of a cytosolic enzyme. When appended to an otherwise cytosolic passenger protein, the bacterial chloramphenicol acetyltransferase (CAT), the prepieces direct the fusion proteins into peroxisomes, demonstrating that they encode sufficient information to act as peroxisomal targeting signals. Deletion analysis of the thiolase B prepiece shows that the first 11 amino acids are sufficient for peroxisomal targeting. We conclude that we have identified a novel PTS that functions at amino-terminal or internal locations and is distinct from the C-terminal PTS. These results imply the existence of two different routes for targeting proteins into the peroxisomal matrix.     

Effect of mutation of C-terminal and heme binding region of Arabidopsis catalase on the import to peroxisomes

We evaluated the import of Arabidopsis catalase to peroxisomes under homogenous transient expression. The amino acids at ?11 to ?4 from the C-terminus are necessary for catalase import. The results are in agreement with the previous work under stable expression. We first demonstrate that heme-binding sites are important for peroxisomal import, suggesting the importance of catalase folding.

Abbreviations: AtCat: Arabidopsis catalase; PTS: peroxisomal targeting signal; PEX: Peroxin     

PTS1-independent targeting of isocitrate lyase to peroxisomes requires the PTS1 receptor Pex5p

The targeting of castor bean isocitrate lyase to peroxisomes was studied by expression in the heterologous host Saccharomyces cerevisae from which the endogenous ICL1 gene had been removed by gene disruption. Peroxisomal import of ICL was dependent upon the PTS1 receptor Pex5p and was lost by deletion of the last three amino acids, Ala-Arg-Met. However, removal of an additional 16 amino acids restored the ability of this truncated ICL to be targeted to peroxisomes and this import activity, like that of the full-length protein, was dependent upon Pex5p. The ability of peptides corresponding to the carboxyl terminal ends of wild-type and Delta 3 and Delta 19 mutants of ICL to interact with the PTS1-binding portion of Pex5p from humans, plants and yeast was determined using the yeast two-hybrid system. The peptide corresponding to wild-type ICL interacted with all three Pex5p proteins to differing extents, but neither mutant could interact with Pex5p from any species. Thus, ICL can be targeted to peroxisomes in a Pex5p-dependent but PTS1-independent fashion. These results help to clarify the contradictory published data about the requirement of the PTS1 signal for ICL targeting.     

Carboxyl-terminal consensus Ser-Lys-Leu-related tripeptide of peroxisomal proteins functions in vitro as a minimal peroxisome-targeting signal.

The minimal sequence requirement for a peroxisome-targeting signal was investigated using an in vitro import system. Carboxyl-terminal sequences Ser-Lys-Leu (SKL) and Leu-Gln-Ser-Lys-Leu (LQSKL) of acyl-CoA oxidase (AOX) directed to peroxisomes the fused proteins with import-incompetent forms of AOX and catalase that had been truncated, implying that the SKL tripeptide functions as a targeting signal. Elimination of the entire SKL sequence or deletion of any 1 or 2 amino acids in the sequence abolished the import activity of AOX. Substitution of alanine for serine did not affect the import activity. Topogenic activity was retained when lysine was mutated to either arginine or histidine, whereas mutation to glutamic acid completely abolished the activity. A synthetic peptide comprising the carboxyl-terminal 10 amino acid residues of AOX inhibited the import of the authentic AOX polypeptide, whereas other peptides in which SKL was mutated, deleted, or internally located were not effective. The uptake of AOX was little affected by the peptide with an amidated alpha-carboxyl group. These results strongly suggest that the carboxyl-terminal SKL motif sequence (Ser/Ala)-(Lys/Arg/His)-Leu functions as a topogenic signal in translocation of proteins into peroxisomes, requiring the whole tripeptide sequence with a free alpha-COOH group at the carboxyl terminus.     

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.     

Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p

PEX5 functions as an import receptor for proteins with the type-1 peroxisomal targeting signal (PTS1). Although PEX5 is not involved in the import of PTS2-targeted proteins in yeast, it is essential for PTS2 protein import in mammalian cells. Human cells generate two isoforms of PEX5 through alternative splicing, PEX5S and PEX5L, and PEX5L contains an additional insert 37 amino acids long. Only one isoform, PEX5L, is involved in PTS2 protein import, and PEX5L physically interacts with PEX7, the import receptor for PTS2-containing proteins. In this report we map the regions of human PEX5L involved in PTS2 protein import, PEX7 interaction, and targeting to peroxisomes. These studies revealed that amino acids 1-230 of PEX5L are required for PTS2 protein import, amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for targeting to peroxisomes. We also identified a 21-amino acid-long peptide motif of PEX5L, amino acids 209-229, that overlaps the regions sufficient for full PTS2 rescue activity and PEX7 interaction and is shared by Saccharomyces cerevisiae Pex18p and Pex21p, two yeast peroxins that act only in PTS2 protein import in yeast. A mutation in PEX5 that changes a conserved serine of this motif abrogates PTS2 protein import in mammalian cells and reduces the interaction of PEX5L and PEX7 in vitro. This peptide motif also lies within regions of Pex18p and Pex21p that interact with yeast PEX7. Based on these and other results, we propose that mammalian PEX5L may have acquired some of the functions that yeast Pex18p and/or Pex21p perform in PTS2 protein import. This hypothesis may explain the essential role of PEX5L in PTS2 protein import in mammalian cells and its lack of importance for PTS2 protein import in yeast.     

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

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

Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy

In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurrs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide. Received: 4 March 1998 / Accepted: 2 July 1998     

Peroxisomal targeting signals in green algae

Peroxisomal enzymatic proteins contain targeting signals (PTS) to enable their import into peroxisomes. These targeting signals have been identified as PTS1 and PTS2 in mammalian, yeast, and higher plant cells; however, no PTS2-like amino acid sequences have been observed in enzymes from the genome database of Cyanidiochyzon merolae (Bangiophyceae), a primitive red algae. In studies on the evolution of PTS, it is important to know when their sequences came to be the peroxisomal targeting signals for all living organisms. To this end, we identified a number of genes in the genome database of the green algae Chlamydomonas reinhardtii, which contains amino acid sequences similar to those found in plant PTS. In order to determine whether these sequences function as PTS in green algae, we expressed modified green fluorescent proteins (GFP) fused to these putative PTS peptides under the cauliflower mosaic virus 35S promoter. To confirm whether granular structures containing GFP–PTS fusion proteins accumulated in the peroxisomes of Closterium ehrenbergii, we observed these cells after the peroxisomes were stained with 3, 3′-diaminobenzidine. Our results confirm that the GFP–PTS fusion proteins indeed accumulated in the peroxisomes of these green algae. These findings suggest that the peroxisomal transport system for PTS1 and PTS2 is conserved in green algal cells and that our fusion proteins can be used to visualize peroxisomes in live cells.     

Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes

Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom.     

Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence

We have identified a novel peroxisomal targeting sequence (PTS) at the extreme COOH terminus of human catalase. The last four amino acids of this protein (-KANL) are necessary and sufficient to effect targeting to peroxisomes in both human fibroblasts and Saccharomyces cerevisiae, when appended to the COOH terminus of the reporter protein, chloramphenicol acetyl transferase. However, this PTS differs from the extensive family of COOH-terminal PTS tripeptides collectively termed PTS1 in two major aspects. First, the presence of the uncharged amino acid, asparagine, at the penultimate residue of the human catalase PTS is highly unusual, in that a basic residue at this position has been previously found to be a common and critical feature of PTS1 signals. Nonetheless, this asparagine residue appears to constitute an important component of the catalase PTS, in that replacement with aspartate abolished peroxisomal targeting (as did deletion of the COOH-terminal four residues). Second, the human catalase PTS comprises more than the COOH-terminal three amino acids, in that COOH-terminal-ANL cannot functionally replace the PTS1 signal-SKL in targeting a chloramphenicol acetyl transferase fusion protein to peroxisomes. The critical nature of the fourth residue from the COOH terminus of the catalase PTS (lysine) is emphasized by the fact that substitution of this residue with a variety of other amino acids abolished or reduced peroxisomal targeting. Targeting was not reduced when this lysine was replaced with arginine, suggesting that a basic amino acid at this position is required for maximal functional activity of this PTS. In spite of these unusual features, human catalase is sorted by the PTS1 pathway, both in yeast and human cells. Disruption of the PAS10 gene encoding the S. cerevisiae PTS1 receptor resulted in a cytosolic location of chloramphenicol acetyl transferase appended with the human catalase PTS, as did expression of this protein in cells from a neonatal adrenoleukodystrophy patient specifically defective in PTS1 import. Furthermore, through the use of the two-hybrid system, it was demonstrated that both the PAS10 gene product (Pas10p) and the human PTS1 receptor can interact with the COOH-terminal region of human catalase, but that this interaction is abolished by substitutions at the penultimate residue (asparagine-to- aspartate) and at the fourth residue from the COOH terminus (lysine-to-glycine) which abolish PTS functionality. We have found no evidence of additional targeting information elsewhere in the human catalase protein. An internal tripeptide (-SHL-, which conforms to the mammalian PTS1 consensus) located nine to eleven residues from the COOH terminus has been excluded as a functional PTS. Additionally, in contrast to the situation for S. cerevisiae catalase A, which contains an internal PTS in addition to a COOH-terminal PTS1, human catalase lacks such a redundant PTS, as evidenced by the exclusive cytosolic location of human catalase mutated in the COOH-terminal PTS. Consistent with this species difference, fusions between catalase A and human catalase which include the catalase A internal PTS are targeted, at least in part, to peroxisomes regardless of whether the COOH-terminal human catalase PTS is intact.     

Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein

The ICL1 gene encoding isocitrate lyase was cloned from the dimorphic fungus Yarrowia lipolytica by complementation of a mutation (acuA3) in the structural gene of isocitrate lyase of Escherichia coli. The open reading frame of ICL1 is 1668 by long and contains no introns in contrast to currently sequenced genes from other filamentous fungi. The ICL1 gene encodes a deduced protein of 555 amino acids with a molecular weight of 62 kDa, which fits the observed size of the purified monomer of isocitrate lyase from Y. lipolytica. Comparison of the protein sequence with those of known pro- and eukaryotic isocitrate lyases revealed a high degree of homology among these enzymes. The isocitrate lyase of Y. lipolytica is more similar to those from Candida tropicalis and filamentous fungi than to Sacharomyces cerevisiae. This enzyme of Y. lipolytica has the putative glyoxysomal targeting signal S-K-L at the carboxy-terminus. It contains a partial repeat which is typical for eukaryotic isocitrate lyases but which is absent from the E. coli enzyme. Surprisingly, deletion of the ICL1 gene from the genome not only inhibits the utilization of acetate, ethanol, and fatty acids, but also reduces the growth rate on glucose.     

Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-- >Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg- mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.     

The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with the analogous native domain for peroxisomal import of PTS1-terminated proteins in yeast

In the yeast Saccharomyces cerevisiae, beta-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5-TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5delta mutant phenotype. Expression of the Pex5p chimaeras in pex5delta mutant cells restored peroxisomal import of PTS1-terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5-TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5delta cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL.     

Protein quaternary structure and expression levels contribute to peroxisomal-targeting-sequence-1-mediated peroxisomal import of human soluble epoxide hydrolase

The peroxisomal targeting sequence 1 (PTS1) is a consensus tripeptide 1 (S/C/A)(K/R/H)(L/M) that is found at the C-terminus of most peroxisomal proteins. However, the only known mammalian protein containing a terminal methionine PTS1 (SKM), human soluble epoxide hydrolase (hsEH), shows both peroxisomal and cytosolic localizations in vivo. Mechanisms regulating the subcellular localization of hsEH thus remain unclear. Here we utilized green fluorescent protein-hsEH fusion constructs to study the peroxisomal targeting of hsEH in transiently and stably transfected Chinese hamster ovary cells. Our results suggest that the peroxisomal import of hsEH is regulated by three factors. First, we show that SKM is required, but not sufficient, for peroxisomal import. Second, by manipulating protein expression levels, we show that SKM mediates peroxisomal import of wild-type hsEH only when expression levels are high. Third, we show that amino acid modifications that decrease subunit oligomerization and presumably enhance accessibility of the SKM motif confer peroxisomal targeting even at low protein expression levels. We conclude that, in hsEH, SKM is a necessary but inefficient and context-dependent PTS1. Peroxisomal import occurs when expression levels are high or when the SKM motif is accessible. These results provide a mechanistic basis for understanding the cell-specific and tissue-specific localization of hsEH in vivo.     

Import of the peroxisomal targeting signal type 2 protein 3-ketoacyl-coenzyme a thiolase into glyoxysomes

Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.