Identification and characterization of the human peroxin PEX3.

The biogenesis of peroxisomes requires the interaction of several peroxins, encoded by PEX genes and is well conserved between yeast and humans. We have cloned the human cDNA of PEX3 based on its homology to different yeast PEX3 genes. The deduced peroxin HsPEX3 is a peroxisomal membrane protein with a calculated molecular mass of 42.1 kDa. We created N- and C-terminal tagged PEX3 to assay its topology at the peroxisomal membrane by immunofluorescence microscopy. Our results and the one predicted transmembrane spanning region are in line with the assumption that H sPEX3 is an integral peroxisomal membrane protein with the N-terminus inside the peroxisome and the C-terminus facing the cytoplasm. The farnesylated peroxisomal membrane protein PEX19 interacts with HsPEX3 in a mammalian two-hybrid assay in human fibroblasts. The physical interaction could be confirmed by coimmunoprecipitation of the two in vitro transcribed and translated proteins. To address the targeting of PEX3 to the peroxisomal membrane, the expression of different N- and C-terminal PEX3 truncations fused to green fluorescent protein (GFP) was investigated in human fibroblasts. The N-terminal 33 amino acids of PEX3 were necessary and sufficient to direct the reporter protein GFP to peroxisomes and seemed to be integrated into the peroxisomal membrane. The expression of a 1-16 PEX3-GFP fusion protein did not result in a peroxisomal localization, but interestingly, this and several other truncated PEX3 fusion proteins were also localized to tubular and/or vesicular structures representing mitochondria.     

Sorting of peroxisomal membrane protein PMP47 from Candida boidinii into peroxisomal membranes of Saccharomyces cerevisiae

A gene encoding PMP47, a peroxisomal integral membrane protein of the methylotrophic yeast Candida boidinii, was isolated from a genomic library. DNA sequencing of PMP47 revealed an open reading frame of 1269 base pairs capable of encoding a protein of 46,873 Da. At least two membrane-spanning regions in the protein are predicted from the sequence. Since the 3 amino acids at the carboxyl terminus are -AKE, PMP47 lacks a typical peroxisomal sorting signal. No significant similarities in primary structure between PMP47 and known proteins were observed, including PMP70, a rat peroxisomal membrane protein whose sequence has recently been reported (Kamijo, K., Taketani, S., Yokota, S., Osumi, T., and Hashimoto, T. (1990). J. Biol. Chem. 265, 4534-4540). In order to study the import and assembly of PMP47 into peroxisomes by genetic approaches, the gene was expressed in the yeast Saccharomyces cerevisiae. When PMP47 was expressed in cells grown on oleic acid to induce peroxisomes, the protein was observed exclusively in peroxisomes as determined by marker enzyme analysis of organelle fractions. Most of the PMP47 co-purified with the endogenous peroxisomal membrane proteins on isopycnic sucrose gradients. Either in the native host or when expressed in S. cerevisiae, PMP47 was not extractable from peroxisomal membranes by sodium carbonate at pH 11, indicating an integral membrane association. These results indicate that PMP47 is competent for sorting to and assembling into peroxisomal membranes in S. cerevisiae.     

Peroxisomal protein import is conserved between yeast, plants, insects and mammals.

We have previously demonstrated that firefly luciferase can be imported into peroxisomes of both insect and mammalian cells. To determine whether the process of protein transport into the peroxisome is functionally similar in more widely divergent eukaryotes, the cDNA encoding firefly luciferase was expressed in both yeast and plant cells. Luciferase was translocated into peroxisomes in each type of organism. Experiments were also performed to determine whether a yeast peroxisomal protein could be transported to peroxisomes in mammalian cells. We observed that a C-terminal segment of the yeast (Candida boidinii) peroxisomal protein PMP20 could act as a peroxisomal targeting signal in mammalian cells. These results suggest that at least one mechanism of protein translocation into peroxisomes has been conserved throughout eukaryotic evolution.     

The carboxyl-terminal tripeptide serine-lysine-leucine of firefly luciferase is necessary but not sufficient for peroxisomal import in yeast.

Firefly luciferase is imported into peroxisomes in insects, mammals, plants, and yeast, which implies that the mechanism of protein translocation into peroxisomes has been conserved during eukaryotic evolution. The carboxyl-terminal tripeptide serine-lysine-leucine in luciferase acts as a peroxisomal import signal in mammalian cells. We have investigated whether this tripeptide is also involved in translocation of firefly luciferase into peroxisomes in yeast (Saccharomyces cerevisiae). We show by gene fusion experiments that the carboxyl-terminal 104 amino acids of luciferase can direct a heterologous protein to yeast peroxisomes. Luciferase mutant proteins were tested for their ability to be imported into yeast peroxisomes in vivo. We demonstrate that mutations in the carboxyl-terminal serine-lysine-leucine tripeptide abolish translocation of the protein into yeast peroxisomes. However, when a passenger protein was tagged at its carboxyl terminus with this tripeptide the fusion protein did not go to peroxisomes. These results indicate that, in yeast, the tripeptide is necessary but not sufficient for peroxisomal import.     

Targeting elements in the amino-terminal part direct the human 70-kDa peroxisomal integral membrane protein (PMP70) to peroxisomes.

Peroxisomes are multipurpose organelles present in nearly all eukaryotic cells. All peroxisomale matrix and membrane proteins are synthesized in the cytoplasm. While a clear picture of the basic targeting mechanisms for peroxisomal matrix proteins has emerged over the past years, the targeting processes for peroxisomal membrane proteins are poorly understood. The 70-kDa peroxisomal integral membrane protein (PMP70) is one of the proteins located in the human peroxisome membrane. PMP70 belongs to the family of ATP-binding cassette (ABC) transporter proteins. It consists of six transmembrane domains and an ATP-binding fold in the cytosol. Here we describe that efficient peroxisomal targeting of human PMP70 depends on three targeting elements in the amino-terminal protein region, namely amino acids 61 to 80 located in the cytosol as well as the first and second transmembrane domains. Furthermore, peroxin 19 (PEX19) interactions are not required for targeting human PMP70 to peroxisomes. PEX19 does not specifically bind to the targeting elements of human PMP70.     

Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.     

An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320- 322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47- dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.     

Peroxisome targeting of porcine 17beta-hydroxysteroid dehydrogenase type IV/D-specific multifunctional protein 2 is mediated by its C-terminal tripeptide AKI

The product of the porcine HSD17B4 gene is a peroxisomal 80 kDa polypeptide containing three functionally distinct domains. The N-terminal part reveals activities of 17beta-estradiol dehydrogenase type IV and D-specific 3-hydroxyacyl CoA dehydrogenase, the central part shows D-specific hydratase activity with straight and 2-methyl-branched 2-enoyl-CoAs. The C-terminal part is similar to sterol carrier protein 2. The 80 kDa polypeptide chain ends with the tripeptide AKI, which resembles the motif SKL, the first identified peroxisome targeting signal PTS1. So far AKI, although being similar to the consensus sequence PTS1, has neither been reported to be present in mammalian peroxisomal proteins, nor has it been shown to be functional. We investigated whether the HSD17B4 gene product is targeted to peroxisomes by this C-terminal motif. Recombinant human PTS1 binding protein Pex5p interacted with the bacterially expressed C-terminal domain of the HSD17B4 gene product. Binding was competitively blocked by a SKL-containing peptide. Recombinant deletion mutants of the C-terminal domain lacking 3, 6, and 14 amino acids and presenting KDY, MIL, and IML, respectively, at their C-termini did not interact with Pex5p. The wild-type protein and mutants were also transiently expressed in the HEK 293 cells. Immunofluorescence analysis with polyclonal antibodies against the C-terminal domain showed a typical punctate peroxisomal staining pattern upon wild-type transfection, whereas all mutant proteins localized in the cytoplasm. Therefore, AKI is a functional PTS1 signal in mammals and the peroxisome targeting of the HSD17B4 gene product is mediated by Pex5p.     

The carboxyl-terminal tripeptide Ala-Lys-Ile is essential for targeting Candida tropicalis trifunctional enzyme to yeast peroxisomes.

The gene encoding Candida tropicalis peroxisomal trifunctional enzyme, hydratase-dehydrogenase-epimerase (HDE), was expressed in both Candida albicans and Saccharomyces cerevisiae. The cellular location of HDE was determined by subcellular fractionation followed by Western blot analysis of peroxisomal and cytosolic fractions using antiserum specific for HDE. HDE was found to be exclusively targeted to and imported into peroxisomes in both heterologous expression systems. Deletion and mutational analyses were used to determine the regions within HDE which are essential for its targeting to peroxisomes. Deletion of a carboxyl-terminal tripeptide Ala-Lys-Ile completely abolished targeting of HDE to peroxisomes, whereas large internal deletions of HDE (amino acids 38-353 or 395-731) had no effect on HDE targeting to peroxisomes in either yeast. This tripeptide is similar to, but distinct from, other tripeptide peroxisomal targeting sequences (PTSs) as identified in peroxisomal firefly luciferase and four mammalian peroxisomal proteins. Substitutions within the carboxyl-terminal tripeptide (Ala----Gly and Lys----Gln) supported targeting of HDE to peroxisomes of C. albicans but not of S. cerevisiae. This is the first detailed analysis of the peroxisomal targeting signal in a yeast peroxisomal protein.     

Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.     

The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein superfamily

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major integral membrane proteins of rat liver peroxisomes. cDNA clones for PMP70 were isolated and sequenced. The predicted amino acid sequence (659 amino acid residues) revealed that the carboxyl-terminal region of PMP70 has strong sequence similarities to a group of ATP-binding proteins such as MalK and Mdr. These proteins form a superfamily and are involved in various biological processes including membrane transport. Limited protease treatment of peroxisomes showed that the ATP-binding domain of PMP70 is exposed to the cytosol. The hydropathy profile, in comparison with those of several other members of the ATP-binding protein superfamily, suggests that PMP70 is a transmembrane protein possibly forming a channel. Based on these results, we propose that PMP70 is involved in active transport across the peroxisomal membrane.     

Topogenesis of peroxisomal membrane protein requires a short, positively charged intervening-loop sequence and flanking hydrophobic segments. study using human membrane protein PMP34

Human 34-kDa peroxisomal membrane protein (PMP34) consisting of 307 amino acids was previously identified as an ortholog of, or a similar protein (with 27% identity) to the, 423-amino acid-long PMP47 of the yeast Candida boidinii. We investigated membrane topogenesis of PMP34 with six putative transmembrane segments, as a model peroxisomal membrane protein. PMP34 was characterized as an integral membrane protein of peroxisomes. Transmembrane topology of PMP34 was determined by differential permeabilization and immunofluorescent staining of HeLa cells ectopically expressing PMP34 as well as of Chinese hamster ovary-K1 expressing epitope-tagged PMP34. As opposed to PMP47, PMP34 was found to expose its N- and C-terminal parts to the cytosol. Various deletion variants of PMP34 and their fusion proteins with green fluorescent protein were expressed in Chinese hamster ovary-K1 and were verified with respect to intracellular localization. The loop region between transmembrane segments 4 and 5 was required for the peroxisome-targeting activity, in which Ala substitution for basic residues abrogated the activity. Three hydrophobic transmembrane segments linked in a flanking region of the basic loop were essential for integration of PMP34 to peroxisome membranes. Therefore, it is evident that the intervening basic loop plus three transmembrane segments of PMP34 function as a peroxisomal targeting and topogenic signal.     

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.     

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.     

Two genes encode the major membrane-associated protein of methanol-induced peroxisomes from Candida boidinii

A massive proliferation of peroxisomes occurs in the yeast Candida boidinii when methanol is utilized as the sole carbon source; these peroxisomes contain the enzymes which catalyze the initial steps of methanol utilization. The most abundant peroxisomal membrane-associated protein has an apparent molecular mass of 20 kDa and is termed PMP20. We report the isolation of two genes that encode very similar forms of PMP20; this is the first report of genes that encode proteins associated with peroxisomal membranes. Southern analysis demonstrates that the two genes are on different loci, although there are several homologous regions of both 5'- and 3'-untranslated sequence. One of the areas of 5' homology is within the untranslated region of the mRNA. Within the coding region there are 35 base differences between the two genes that are reflected in only five amino acid differences. The mRNAs representing both genes of PMP20 are induced in cells grown in methanol-containing medium and are below detection in cells grown in glucose. S1 nuclease protection analysis indicates that there is a 2.5-fold difference in mRNA expression between the two genes when induced. The predicted sequences of both PMP20 genes show the absence of a cleaved amino-terminal leader sequence and the presence of only 1 cysteine residue. In agreement with previous biochemical data suggesting a peripheral association of this protein with the membrane (Goodman, J. M., Maher, J., Silver, P. A., Pacifico, A., and Sanders, D. (1986) J. Biol. Chem. 261, 3464-3468), there are no obvious membrane spanning regions predicted in the sequences. Both PMP20 gene products contain the carboxyl-terminal sequence AKL, similar to the putative SKL peroxisomal sorting sequence (Gould, S. J., Keller, G.-A., and Subramani, S. (1988) J. Cell Biol. 107, 897-905).     

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.     

Import of stably folded proteins into peroxisomes.

By virtue of their synthesis in the cytoplasm, proteins destined for import into peroxisomes are obliged to traverse the single membrane of this organelle. Because the targeting signal for most peroxisomal matrix proteins is a carboxy-terminal tripeptide sequence (SKL or its variants), these proteins must remain import competent until their translation is complete. We sought to determine whether stably folded proteins were substrates for peroxisomal import. Prefolded proteins stabilized with disulfide bonds and chemical cross-linkers were shown to be substrates for peroxisomal import, as were mature folded and disulfide-bonded IgG molecules containing the peroxisomal targeting signal. In addition, colloidal gold particles conjugated to proteins bearing the peroxisomal targeting signal were translocated into the peroxisomal matrix. These results support the concept that proteins may fold in the mammalian cytosol, before their import into the peroxisome, and that protein unfolding is not a prerequisite for peroxisomal import.     

Identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle

Peroxisomes are unimembrane, respiratory organelles of the cell. Transport of cellular proteins to the peroxisomal matrix requires a type 1 peroxisomal targeting signal (PTS1) which essentially constitutes a tripeptide from the consensus sequence S/T/A/G/C/N-K/R/H-L/I/V/M/A/F/Y. Although PTS-containing proteins have been identified in eukaryotes, prokaryotes, and parasites, viral proteins with such signals have not been identified so far. We report here the first instance of a virus, the rotavirus, which causes infantile diarrhea worldwide, containing a functional C-terminal PTS1 in one of its proteins (VP4). Analysis of 153 rotavirus VP4-deduced amino acid sequences identified five groups of conserved C-terminal PTS1 tripeptide sequences (SKL, CKL, GKL, CRL, and CRI), of which CRL is represented in approximately 62% of the sequences. Infection of cells by a CRL-containing representative rotavirus (SA11 strain) and confocal immunofluorescence analysis revealed colocalization of VP4 with peroxisomal markers and morphological changes of peroxisomes. Further, transient cellular expression of green fluorescent protein (GFP)-fused VP4CRL resulted in transport of VP4 to peroxisomes, whereas the chimera lacking the PTS1 signal, GFP-VP4DeltaCRL, resulted in diffuse cytoplasmic staining, suggesting a CRL-dependent targeting of the protein. The present study therefore demonstrates hitherto unreported organelle involvement, specifically of the peroxisomes, in rotaviral infections as demonstrated by using the SA11 strain of rotavirus and opens a new line of investigation toward understanding viral pathogenesis and disease mechanisms.     

Transport of microinjected proteins into peroxisomes of mammalian cells: inability of Zellweger cell lines to import proteins with the SKL tripeptide peroxisomal targeting signal.

Previous work has shown that the firefly (Photinus pyralis) luciferase contains a C-terminal peroxisomal targeting signal consisting of the tripeptide Ser-Lys-Leu. This report describes the microinjection of two proteins, (i) luciferase and (ii) albumin conjugated to a peptide ending in the sequence Ser-Lys-Leu, into mammalian cells grown in tissue culture. Following microinjection, incubation of the cells at 37 degrees C resulted in peroxisomal transport of these exogenous proteins into catalase-containing vesicles. The translocation was both time and temperature dependent. The transport could be inhibited by coinjection of synthetic peptides bearing various peroxisomal targeting signal motifs. These proteins could be transported into peroxisomes in normal human fibroblast cell lines but not in cell lines derived from patients with Zellweger syndrome. These results demonstrate that microinjection of peroxisomal proteins yields an authentic in vivo system with which to study peroxisomal transport. Furthermore, these results reveal that the process of peroxisomal transport does not involve irreversible modification of the protein, that artificial hybrid substrates can be transported and used as tools to study peroxisomal transport, and that the defect in Zellweger syndrome is indeed the inability to transport proteins containing the Ser-Lys-Leu targeting signal into the peroxisomal lumen.     

Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins

As part of an effort to understand how proteins are imported into the peroxisome, we have sought to identify the peroxisomal targeting signals in four unrelated peroxisomal proteins: human catalase, rat hydratase:dehydrogenase, pig D-amino acid oxidase, and rat acyl-CoA oxidase. Using gene fusion experiments, we have identified a region of each protein that can direct heterologous proteins to peroxisomes. In each case, the peroxisomal targeting signal is contained at or near the carboxy terminus of the protein. For catalase, the peroxisomal targeting signal is located within the COOH-terminal 27 amino acids of the protein. For hydratase:dehydrogenase, D-amino acid oxidase, and acyl-CoA oxidase, the targeting signals are located within the carboxy-terminal 15, 14, and 15 amino acids, respectively. A tripeptide of the sequence Ser-Lys/His-Leu is present in each of these targeting signals as well as in the peroxisomal targeting signal identified in firefly luciferase (Gould, S.J., G.-A. Keller, and S. Subramani. 1987. J. Cell Biol. 105:2923-2931). When the peroxisomal targeting signal of the hydratase:dehydrogenase is mutated so that the Ser-Lys-Leu tripeptide is converted to Ser-Asn-Leu, it can no longer direct proteins to peroxisomes. We suggest that this tripeptide is an essential element of at least one class of peroxisomal targeting signals.