©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of the Putative Human Peroxisomal C-terminal Targeting Signal Import Receptor (*)

(Received for publication, January 19, 1995; and in revised form, February 7, 1995)

Marc Fransen (1) Chantal Brees (1) Eveline Baumgart (1) (3)(§) Johannes C. T. Vanhooren (1) Myriam Baes (2) Guy P. Mannaerts (1)(¶) Paul P. Van Veldhoven (1)(¶)

From the  (1)Afdeling Farmakologie and (2)Afdeling Klinische Chemie, Campus Gasthuisberg, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium, the (3)University of Heidelberg, Institute for Anatomy and Cell Biology II, INF 307, D-69120 Heidelberg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To identify proteins interacting with the C-terminal peroxisomal targeting signal (PTS1), we screened a human liver cDNA library by means of a Saccharomyces cerevisiae genetic system, known as the two-hybrid system. We isolated a cDNA encoding a protein that specifically bound the PTS1 topogenic signal in the intact yeast cell but also in vitro after bacterial expression and purification. Sequence analysis of the full-length cDNA revealed the presence of an open reading frame encoding a 70-kDa polypeptide that belongs to the tetratricopeptide repeat family and that is homologous to the PAS8 and PAS10 gene products, which are required for the formation of normal peroxisomes in yeast. Subcellular fractionation of human liver and immunofluorescence studies on HepG(2) cells demonstrated that this PTS1-binding protein is present exclusively in peroxisomes and that the PTS1-binding domain is located to the cytosolic side of the peroxisomal membrane. All available evidence indicates that the PTS1-binding protein is part of the peroxisomal protein import machinery and most probably is the long sought after human PTS1 import receptor.


INTRODUCTION

Peroxisomes are single membrane-bound organelles found in almost all eukaryotic cells and involved in a variety of metabolic functions (1) . Peroxisomal proteins are synthesized on free polyribosomes in the cytosol and are post-translationally imported into pre-existing peroxisomes(2) . Most, but not all, matrix proteins contain a C-terminal tripeptide (serine-lysine-leucine-COOH or a conservative variant) that acts as a peroxisomal targeting signal (PTS1)(^1)(3, 4) . In contrast to the well defined characterization of the PTS1 topogenic signal, little is known about the mechanism of import including the recognition of the targeting signal. Complementation studies on yeast mutants unable to form normal peroxisomes have shown that at least 18 gene products are involved in peroxisomal protein import and biogenesis in this organism (5, 6) . Although several of the genes have been cloned, the exact function and, in most cases, also the subcellular localization of the corresponding proteins remain unknown(5) . Complementation studies on cell lines from patients with generalized peroxisomal disorders (e.g. the Zellweger syndrome), believed to be peroxisomal protein translocation defects, have indicated that also in the human at least nine gene products are required for peroxisome biogenesis(7) . Only two of these proteins have been identified: a 35-kDa peroxisomal membrane protein, which was revealed by functional complementation analysis(8) , and a previously identified 70-kDa peroxisomal membrane protein, belonging to the ATP-binding cassette transporter family, which proved to be mutated in several Zellweger patients(9) . The exact function of these proteins as well remains unknown.

In an attempt to identify the human PTS1 import receptor, we utilized the two-hybrid system (10) to screen a human liver cDNA library, subcloned into the Gal4-encoding plasmid pGAD10, with a vector encoding a fusion protein of the Gal4 and the 70 C-terminal amino acids of rat palmitoyl-CoA oxidase, which displays the C-terminal PTS1.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases, modifying enzymes, and factor Xa were purchased from Boehringer Mannheim. The plasmid pMJ125 (11) and the antibody against Mn-superoxide dismutase were kindly provided by Prof. T. Osumi (Nagano) and Prof. J. Metz (Heidelberg), respectively. All the peptides used were made with a peptide synthesizer (9050 Pepsynthesizer, Milligen/Biosearch) as described previously(12) . The two-hybrid system and the Pinpoint protein purification system were purchased from Clontech and Promega, respectively. All oligonucleotide primers were obtained from Pharmacia Biotech Inc.

Plasmids

For construction of the DNA-binding domain/PTS1 hybrid (pFB1), the 1.7-kb BamHI-SalI insert of the rat palmitoyl-CoA oxidase-encoding pMJ125 vector was ligated into the BamHI-SalI-digested Gal4-encoding vector pGBT9. The hybrid construct was verified by sequencing. The control plasmid pFB2 (encoding the same fusion protein as pFB1 but without the C-terminal PTS1) was constructed by self-ligation of the vector after PstI digestion of pFB1.

Screening an Activation Domain cDNA Library

Screening a Gal4 human liver cDNA hybrid library with the two-hybrid system was done according to the manufacturer's instructions (Clontech). Yeast strain HF7c (MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1-HIS3, URA3::(Gal4 17-mers)(3)-CYC1-lacZ) was sequentially transformed with pFB1 and a human liver cDNA library subcloned into the Gal4-encoding plasmid pGAD10 (Clontech) by using the lithium acetate method(13) . Expression of the target protein was checked by Western blotting with an antibody against the 23-kDa subunit of rat palmitoyl-CoA oxidase(14) . To select for colonies containing interacting hybrid plasmids, the transformants were spread on a synthetic minimal dropout agar medium (0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose/appropriate auxotrophic supplements, 2% (w/v) agar) lacking leucine, tryptophan, and histidine but supplemented with 10 mM 3-amino-1,2,4-triazole. beta-Galactosidase activity of HIS3-positive yeast transformants was assayed on nitrocellulose filter replicas according to the manufacturer's instructions. Transformants activating both the HIS3 and beta-galactosidase reporter genes were isolated and three times replated and retested for beta-galactosidase activity. The activation domain plasmid encoding the PTS1-binding protein (pPTS1-BP) was isolated (15) and used to transform Escherichia coli DH5alpha by using the CaCl(2) method(16) . Bacteria transformed with the pPTS1-BP, selected by ampicillin resistance, were identified by restriction enzyme analysis and the isolated plasmid was reintroduced into the yeast strain SFY526 (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, can^r, gal4-542, gal80-538, URA3::GAL1-lacZ) with the DNA-binding domain plasmids pGBT9, pFB1, or pFB2 and tested for beta-galactosidase activity.

Quantitative beta-Galactosidase Assay

Transformed yeast cells (SFY526) were grown on synthetic minimal dropout medium agar plates without leucine and tryptophan. After 3 days, yeast cells were suspended in beta-galactosidase buffer (100 mM sodium-phosphate buffer, pH 7.0, 10 mM KCl, 1 mM MgSO(4)bullet7H(2)O, 37 mM beta-mercaptoethanol) to an optical density of 1.00 at 600 nm. The yeast cells contained in 1 ml of this suspension were pelleted, resuspended in 100 µl of beta-galactosidase buffer, permeabilized by freeze-thawing (liquid nitrogen), and preincubated for 5 min at 30 °C in a shaking water bath (good aeration). After preincubation, 1 mM (final concentration) of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside was added. Transcriptional activation of the beta-galactosidase gene was quantified after 60 min by extracting the water-insoluble blue-indigo reaction product with Me(2)SO and measuring the optical density at 631 nm, as will be published in detail elsewhere.

Expression and Purification of the PTS1-binding Protein in E. coli JM109

The 1.145-kb cDNA insert of pPTS1-BP was excised with BglII and subcloned into the Pinpoint expression vector Xa-1. Insertion of the cDNA into the correct orientation was verified by restriction enzyme analysis with SmaI. Expression of the biotinylated fusion protein and purification by means of monomeric avidin column chromatography was done according to the manufacturer's instructions.

Generation of Antibodies

The purified bacterial biotinylated fusion protein was cleaved with factor Xa according to the manufacturer's instruction. The cleavage was performed overnight at 28 °C at an enzyme to substrate ratio of 1:20 (w/w). The 1.145-kb cDNA-encoded part of the PTS1-binding protein was separated from the biotinylated bacterial part and factor Xa by SDS-gel electrophoresis and injected into rabbits according to standard protocols(17) . Before use the antibodies were affinity purified by using the 1.145-kb cDNA-encoded part of the PTS1-binding protein immobilized on nitrocellulose strips(17) .

Peptide Binding Specificity Assay of the PTS1-binding Protein

One µg of the appropriate peptide was coated (50 mM sodium-carbonate buffer, pH 9.6) overnight onto a microtiter plate. After washing the wells five times with TBST, the wells were blocked with 5% (w/v) Protifar (Nutricia) in TBST for 1 h, and each well was incubated for 1 h with 2.5 µg (250 µl) of purified biotinylated fusion protein diluted in 1% (w/v) Protifar in TBST. After washing the wells five times with TBST, the resulting complex was detected with streptavidin-alkaline phosphatase with p-nitrophenyl phosphate as substrate. The optical density was measured at 405 nm (filter: 690 nm) after 120 min.

Screening a gt11 Human Liver cDNA Library

To obtain the full-length cDNA of the PTS1-binding protein, we hybridized the Plabeled 1.145-kb EcoRI fragment to a gt11 human liver cDNA library (Clontech) according to standard protocols(17) . Positive plaques were isolated and after DNA preparation (Sephaglas Phageprep kit, Pharmacia) the clones were analyzed by PCR according to the manufacturer's instructions (Perkin Elmer). The PCR reactions were performed by using a program with a general profile of: denaturation at 95 °C for 45 s, annealing for 60 s at t(x), and extension for 3 min at 72 °C. The value of t(x) was determined empirically for each pair of primers.

DNA Sequencing

Sequencing was carried out according to the method of Sanger(18) . The 1.145-kb EcoRI insert of the Gal4 plasmid pPTS1-BP was subcloned into pBluescript SK and the cDNA sequence was determined with a T7 sequencing kit (Pharmacia). PCR products of positive phage DNA were purified (Geneclean II kit, Bio101) and cycle sequenced (AmplyCycle Sequencing kit, Perkin Elmer) according to the manufacturer's instructions. The cDNA sequence was obtained using the T7 and T3 sequencing primers for pBluescript, the gt11 forward and reverse primers, and specific oligonucleotide primers. All regions of the cDNA were sequenced at least once in both directions.

Preparation of Subcellular Fractions from Human Liver

Superfluous tissue from a transplantation liver was used. Approval was granted by the Institutional Ethics Committee. The tissue was homogenized and fractionated by differential centrifugation into a nuclear(N), a heavy mitochondrial (M), a light mitochondrial (L), a microsomal (P), and a soluble (S) fraction. The L-fraction, enriched in peroxisomes, was subfractionated by isopycnic centrifugation in an iso-osmotic self-generating Percoll gradient. Subsequently, the Percoll-purified peroxisomal fraction was subfractionated by centrifugation through a discontinuous Nycodenz gradient(19) . All fractions were analyzed for protein and marker enzymes(19) .

SDS-PAGE and Western Blotting

Proteins were separated by SDS-PAGE in ExcelGel SDS (gradient 8-18) precast gels on a horizontal electrophoresis system (Pharmacia). After electrophoresis, the gels were semi-dry transferred to nitrocellulose (0.45 µm; Schleicher & Schuell)(20) . The proteins on the nitrocellulose membranes were briefly visualized by Ponceau S staining(21) . After destaining, the membranes were immunostained as described previously (12) by using the affinity-purified antibody against the 1.145-kb cDNA-encoded part of the PTS1-binding protein.

Immunofluorescence Studies

HepG(2) cells were grown in alpha-minimal essential medium on glass coverslips. The cells were fixed with 4% (w/v) paraformaldehyde/phosphate-buffered saline, and the indirect immunofluorescence microscopy was done as described(22) . Double immunofluorescence studies were performed using the affinity-purified polyclonal rabbit anti-human PTS1 receptor antibody in conjunction with a polyclonal sheep anti-rat catalase antibody or a monoclonal mouse anti-mitochondrial Mn-superoxide dismutase antibody. The primary antibodies were either detected with donkey anti-rabbit FITC-/donkey anti-sheep-Cy3-labeled or goat anti-rabbit-FITC-/goat anti-mouse RITC-labeled secondary antibodies. To selectively permeabilize the plasma membrane and to localize the functional part of the PTS1 receptor to the cytosolic surface of the peroxisomal membrane, double immunofluorescence studies with decreasing Triton X-100 concentrations (1%, 0.5%, 0.1%, 0.05%, 0.01%, and 0.005%, w/v) were performed.


RESULTS AND DISCUSSION

Using the yeast Gal4 two-hybrid system, we screened 1.5 million clones of a human liver cDNA expression library and identified one transformant activating both the HIS3 and beta-galactosidase reporter genes. Reintroduction of the Gal4 plasmid, containing a 1.145-kb cDNA insert encoding the presumed PTS1-binding protein, of this transformant into another yeast strain with other DNA-binding domain plasmids excluded the possibility of a false positive (Table 1, part a). The specific in vivo interaction identified by the two-hybrid approach was also confirmed by an in vitro binding assay (Table 1, part b). Therefore, the PTS1-binding protein was expressed in E. coli and the purified binding protein was incubated with several peptides. The presumed PTS1-binding protein recognized only the peptide containing the PTS1 signal and not other peptides such as those containing an amidated PTS1 or the N-terminal presequence of rat peroxisomal thiolase, which constitutes a peroxisomal targeting signal for this protein(23, 24) . These results demonstrate a direct and specific binding between PTS1 and the part of the putative PTS1 receptor, encoded by the 1.145-kb EcoRI insert of the Gal4 plasmid. The 1.145-kb fragment was used as a probe to obtain the full-length cDNA of the putative receptor.



Sequence analysis of this DNA revealed the presence of an open reading frame encoding a 70,787-Da polypeptide of 639 amino acids (Fig. 1, upper panel). This value was in excellent agreement with that determined by Western blotting using an affinity-purified polyclonal antibody against the 1.145-kb EcoRI-encoded part of the protein (see Fig. 2). A search of data bases revealed that the putative PTS1 receptor belongs to the TPR family and that it has sequence similarity to the PAS8 (33%) and PAS10 (30%) gene products, two other members of this family. The TPR family is characterized by the presence of multiple direct repeats of a degenerate consensus sequence of 34 amino acids (25) (Fig. 1, lowerpanel). The PAS8 and PAS10 gene products are required for the formation of normal peroxisomes in Pichia pastoris and Saccharomyces cerevisiae, respectively, and it has been postulated that these proteins may function as PTS1 import receptors in these yeasts(26, 27) .


Figure 1: Characterization of the PTS1 receptor cDNA. Upperpanel, nucleotide and deduced amino acid sequence of the PTS1 receptor. Nucleotide numbers are given on the left. The Gal4 plasmid contained the 1.145-kb EcoRI cDNA from position 774 to 1919. Both the ATG codons at positions +1 and +7 are putative start codons. The first one is the likely candidate for initiation since the flanking nucleotides follow the Kozak rule(35) . Lower panel, alignment of the nine TPR motifs present in the PTS1 receptor. If an amino acid is present at a given position in four out of the nine repeats, it was termed a consensus residue. The consensus residues in the repeats are presented in bold. Numbers on the left show the positions of the amino acids.




Figure 2: Subcellular distribution of the PTS1-binding protein in human liver. A light mitochondrial fraction, prepared by differential centrifugation and derived from 10 g of human liver, was subfractionated on a self-generating Percoll gradient. The Percoll-purified peroxisomal fractions were pooled and further subfractionated by centrifugation through a discontinuous Nycodenz gradient(19) . The gradient fractions were analyzed for protein (a), glutamate dehydrogenase (mitochondria) (b), acid phosphatase (lysosomes) (c), glucose-6-phosphatase (endoplasmic reticulum) (d), and catalase (peroxisomes) (e). Results are expressed as percentage of total gradient activity or content present in each fraction, numbered on the abscissa. Recoveries for protein and marker enzymes were between 85 and 116%. Samples of the Percoll gradient fractions (2 µl of fractions 1-13) and of the Nycodenz gradient fractions (5 µl of fractions 1-9 and 17-23) were analyzed by immunostaining with the affinity-purified antibody against that part of the PTS1-binding protein encoded by the 1.145-kb cDNA fragment (f). Fractions 10-16 of the Nycodenz gradient were not analyzed by immunostaining because these fractions did not contain proteins. The molecular masses of calibration markers, expressed in kilodaltons, are indicated. The results show that the PTS1-binding protein follows the same distribution pattern as the peroxisomal marker catalase in the gradient.



Subcellular fractionation of human liver by a combination of differential and gradient centrifugation and subsequent immunoblotting of the fractions with the affinity-purified antibody against the 1.145-kb EcoRI-encoded part of the putative PTS1 import receptor demonstrated a reactive protein of 70-kDa that is present exclusively in peroxisomes (Fig. 2). In other fractions not enriched in peroxisomes, including cytosol, no bands were visible. Subfractionation of peroxisomes revealed that the protein is associated with the peroxisomal membrane, from which it could not be extracted by carbonate (pH 11) treatment(28) , indicating that it behaves as an integral membrane protein (data not shown). However, like the PAS8 and PAS10 gene products, the amino acid sequence of the PTS1-binding protein does not show the presence of a putative membrane-spanning region. It has been suggested that the TPR repeats themselves may form alpha-helices resulting in hydrophobic surfaces, which could lead to membrane association(29) . Similar experiments with rat and mouse liver showed that rat and mouse liver peroxisomal membranes also contain a cross-reacting 70-kDa protein (data not shown).

Immunofluorescence studies on HepG2 cells confirmed the exclusive peroxisomal localization of the putative PTS1 receptor (Fig. 3, a and b). Additionally, the PTS1-binding domain could be located to the cytosolic side of the peroxisomal membrane by means of selective permeabilization conditions: permeabilization of the plasma membrane but not of the peroxisomal membrane in the presence of low detergent concentrations stained the putative PTS1 receptor but not catalase, a peroxisomal matrix protein (Fig. 3, c and d). Although the antibody was directed against the TPR motif-containing part of the protein, other proteins of this family did not seem to cross-react (Fig. 3, e and f).


Figure 3: Localization of the putative PTS1 receptor by immunofluorescence microscopy. Double localization of the PTS1 receptor (a) and catalase (b) in HepG2 cells, using permeabilization conditions with 0.05% (w/v) Triton X-100, clearly demonstrates that the cell organelles marked with the PTS1 receptor antibody (FITC filter) can be identified as peroxisomes by superimposition with the appropriate catalase (RITC filter)-positive particles. Double-localization of the PTS1 receptor (c) and catalase (d) using permeabilization with 0.005% (w/v) Triton X-100 shows that the PTS1 receptor is located at the cytosolic side of the peroxisomal membrane. Under these conditions the cell membrane, but not the peroxisomal membrane, is permeabilized since catalase, a peroxisomal matrix protein, is not stained, but the signal for the PTS1 receptor remains clearly visible. The prominent nuclear background staining is due to retention of antibodies because of the weak permeabilization of the cell membrane. Distinct labeling of peroxisomes (PTS1 receptor antibody/FITC filter) (e) and mitochondria (Mn-superoxide dismutase antibody/RITC filter) (f) in double localization experiments demonstrates that there is no cross-reactivity of the PTS1 receptor antibody with possible human analogs of MOM72 or MAS70, other TPR motif proteins.



All available evidence indicates that the protein described in this report is the human PTS1 import receptor. 1) It specifically binds the PTS1 signal when expressed in the intact yeast cell and also in vitro after purification; 2) it is a peroxisomal membrane protein exposed to the cytosol, where peroxisomal proteins are synthesized; 3) it shows homology with the PAS8 and PAS10 gene products, which are required for the formation of normal peroxisomes in yeast. Conversely, our data strongly support the hypothesis that the PAS8 and PAS10 gene products function as PTS1 import receptors in yeast. The PAS8 and PAS10 proteins have been postulated to be PTS1 import receptors, since the corresponding yeast mutants display an import deficiency of peroxisomal PTS1-carrying proteins(26, 27) . In addition, the PAS8 protein also appears to bind the PTS1 sequence in vitro(26) . During the preparation of this manuscript, a report also appeared describing that the PAS10 protein interacts with PTS1 in the two-hybrid system(30) . Thus, the data obtained by others in yeast and our data strongly support the hypothesis that the PAS8 and PAS10 gene products and their human homolog, described in this report, function as PTS1 import receptors. As the PAS8 and PAS10 gene products, the human PTS1 receptor belongs to the TPR protein family, which is involved in mitosis, transcription, splicing, neurogenesis, and stress response(31) . Interestingly, MOM72 and MAS70, the mitochondrial outer membrane import receptors from Neurospora crassa(32) and S. cerevisiae(33) , respectively, also belong to this family. Finally, identification of the mammalian PTS1 receptor will allow for the generation of animals lacking this receptor, and, as a consequence, for the creation of an animal model of Zellweger syndrome, an inherited lethal disease resulting from a defect in the peroxisomal import of PTS1-containing proteins(34) .


FOOTNOTES

*
This work was supported in part by the Belgian ``Fonds voor Geneeskundig Wetenschappelijk Onderzoek'' and the ``Geconcerteerde Onderzoeksacties van de Vlaamse Gemeenschap.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84899[GenBank].

§
Supported in part by the Deutsche Forschungsgemeinschaft.

To whom correspondence should be addressed. Tel.: 32-16-345802; Fax: 32-16-345699.

(^1)
The abbreviations used are: PTS1, C-terminal peroxisomal targeting signal; TPR, tetratricopeptide repeat; Gal4, Gal4 activation domain; Gal4, Gal4 binding domain; TBST, Tris-buffered saline containing 0.05% (v/v) Tween 20; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; RITC, rhodamine isothiocyanate; kb, kilobase pair(s).


ACKNOWLEDGEMENTS

We thank Prof. T. Osumi (Nagano) for the pMJ125 plasmid and Prof. J. Metz (Heidelberg) for the antibody against Mn-superoxide dismutase.


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