(Received for publication, January 19, 1995; and in revised form, February 7, 1995)
From the
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 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.
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)()(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.
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 -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
-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) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84899[GenBank].