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Human Molecular Genetics Pages 1195-1205  

An isoform of Pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes
Introduction
Results
   PEX5 transcripts are alternatively spliced
   Alignment of Pex5p orthologs
   PEX5 expression in patient cell lines
   PEX5L restores both PTS1 and PTS2 import in PBD005 cells
   pPEX5L-N489K restores PTS2 import in PBD005 cells
   pPEX5L-R390ter restores PTS2 import in PBD005 cells
Discussion
   Pex5p is required for both PTS1 and PTS2 protein import
   What is the function of Pex5Lp in PTS2 protein import?
   A molecular explanation for the different patient phenotypes
Materials And Methods
   Cell lines and transfection studies
   Molecular studies of PEX5
   Plasmids
   Antibodies and indirect immunofluorescence
   Characterization of patient cell lines
   Sequence alignment and protein structure analysis
Acknowledgements
References

An isoform of Pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes

An isoform of Pex5p, the human PTS1 receptor, is required for the import of PTS2 proteins into peroxisomes

Nancy Braverman1, Gabriele Dodt2, Stephen J. Gould3,4, David Valle1,5,*

1Departments of Pediatrics and 2Systembiochemie, Ruhr-Universitat, Bochum, Germany and 3Departments of Biological Chemistry and 4Cell Biology and Anatomy and 5Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Received February 13, 1998; Revised and Accepted May 22, 1998

DDBJ/EMBL/GenBank accession no. Z66494

Mutations in the peroxisome targeting signal (PTS) 1 receptor gene, PEX5, are responsible for complementation group (CG) 2 of the peroxisome biogenesis disorders (PBD). Of the two reported patients in this CG, cells from PBD018 (homozygous for the missense mutation N489K) are defective in the import of PTS1 proteins into peroxisomes, as expected. However, cells from PBD005 (homozygous for the nonsense mutation R390ter) are defective in the import of both PTS1 and PTS2 proteins, suggesting that the PTS1 receptor also mediates PTS2-targeted protein import. To investigate this possibility, we characterized PEX5 expression and found that it undergoes alternative splicing, producing two transcripts, one containing (PEX5L) and one lacking (PEX5S) a 111 bp internal exon. Fibroblasts from PBD005 have greatly reduced levels of PEX5 transcript and protein as compared with PBD018. Transfection of PBD005 cells with PEX5S cDNA restores PTS1 but not PTS2 import; transfection with PXR5L cDNA restores both PTS1 and PTS2 protein import. Furthermore, transfection of PBD005 cells with PEX5L cDNAs containing the patient mutations (which are located downstream of the additional exon) restores PTS2 but not PTS1 import. Taken together, these data provide an explanation for the different protein import defects in CG2 patients and show that the long isoform of the Pex5 protein is required for peroxisomal import of PTS2 proteins.

INTRODUCTION

Peroxisomes are single membrane bound organelles present in virtually all eukaryotic cells. Peroxisomal matrix enzymes participate in diverse metabolic activities, including synthesis of plasmalogens, cholesterol and bile acids and [beta]-oxidation of very long chain fatty acids (1-3). The human PBDs are a heterogeneous group of lethal, autosomal recessive diseases characterized by defective import of peroxisomal matrix proteins (4-7). At least 11 PBD CGs have been defined by somatic cell fusion analysis (8,9). The phenotypes of patients in CGs 1-10 comprise a gradient of clinical severity that we refer to as the Zellweger spectrum. Patients with the most severe phenotype, Zellweger syndrome (ZS), have CNS defects, liver dysfunction, renal cysts and die before 1 year of age. Neonatal adrenoleukodystrophy (NALD) and infantile refsum disease (IRD) are similar but progressively milder phenotypes. CG11 is comprised of patients with a distinct phenotype, rhizomelic chondrodysplasia punctata (RCDP), characterized by rhizomelia, cataracts, icthyosis, severe growth and mental retardation.

Peroxisomal matrix proteins are synthesized on free polyribosomes (10) and directed to the organelle by cis-acting PTSs. Most utilize PTS1, a C terminal tripeptide (-SKL or a conservative variant thereof) (11,12). A few, including 3-ketoacyl CoA thiolase (13-15), phytanyl-CoA hydroxylase (16,17), alkyl-dihydroxyacetonephosphate synthase (DHAP-AS) (18) and others (19,20), utilize PTS2, an N-terminal sequence (-R/KLX5Q/HL-). Less well-characterized targeting sequences may be utilized by a few matrix proteins (21-23).

Peroxisome structure and function has been conserved throughout evolution. Assembly of the intact organelle requires the concerted action of a set of peroxisome assembly proteins (peroxins) encoded by PEX genes (24). More than 15 PEX genes have been identified in yeast (24-28). Human orthologs of several of these have been identified by sequence homology (29) or functional complementation (30). Thus far, nine human PEX genes have been described; seven are responsible for known PBD CGs (24,31-37; Warren et al., submitted for publication). PEX5 encodes the PTS1 receptor and is responsible for PBD CG2 (38-40). The PEX5 product (Pex5p) is a 602 amino acid protein with eight tetratricopeptide repeats (TPR); seven occur in tandem in the C-terminal half of the protein and one is in the N-terminal region. TPRs are degenerate motifs of ~34 amino acids thought to be capable of forming interlocking helices that enable protein-protein interactions (41,42). The C-terminal TPR region of Pex5p binds PTS1-containing peptides (38,40,43,44). Pex5p is localized predominantly in the cytosol, although a small amount is present on the cytosolic surface of the peroxisome membrane (38-40). This bimodal distribution at steady-state reflects a dynamic distribution of Pex5p, which cycles between the cytosol and the peroxisome membrane (45). These observations suggest that this receptor binds newly synthesized PTS1 proteins in the cytosol and transports them to the peroxisome for subsequent translocation. Pex5p interacts physically with other peroxins in the cytosol (Pex6p) (46) and at the peroxisomal membrane (Pex13p and Pex14p) (25,26,47-49). Mutations in several PEX genes alter the distribution and/or stability of Pex5p (45).

PEX7 encodes the PTS2 receptor and is the gene responsible for classical RCDP (PBD CG11) (31-33). Yeast and human cells with PEX7 mutations have a specific defect in the import of PTS2, but not PTS1, targeted proteins (6,7,50,51). Pex7p has multiple WD40 motifs, degenerate 40 amino acid sequences which also function in protein-protein interactions, including some with TPR proteins (41,52-54). Pex7p binds PTS2-containing peptides in vitro and in the two-hybrid system (55,56). N-terminal epitope-tagged forms of Pex7p are functional and predominantly cytosolic (31,50). This suggests that, like Pex5p, Pex7p associates with its ligands (PTS2 proteins) in the cytosol and transports them to the peroxisome. Conversely, Zhang et al. found that C-terminal epitope-tagged yeast Pex7p with partially impaired function was mainly intraperoxisomal (51) and identified a putative new N-terminal PTS in Pex7p (56).

Despite the overall conservation of the mechanism of peroxisomal protein import among eukaryotes (57), some differences between various species have been observed. Most notable is the participation of two genes (PEX9 and PEX16) in Yarrowia lipolytica which do not have counterparts in Saccharomyces cerevisiae (27,58). Another species-specific distinction is the apparent requirement for Pex5p in both the PTS1- and PTS2-mediated import pathways in mammalian cells. In yeast, PEX5 mutants (including deletion mutants) are defective only in the import of PTS1 proteins (59-63). One of the two human PEX5 mutant patients (PBD018, with an NALD phenotype) has the expected isolated defect in PTS1; the other (PBD005, with a ZS phenotype) has defective import of both PTS1 and PTS2 proteins (38,39). Recognition of this double import defect led to the suggestion that Pex5p may somehow also function in PTS2-mediated import (39,64). Similarly, Otera et al. (65) and Tsukamoto et al. (66) have recently described Chinese hamster ovary (CHO) cell mutants with defects in both PTS1- and PTS2-mediated import resulting from PEX5 mutations. To further explore the possible role of Pex5p in PTS2-mediated import, we performed additional studies on PEX5 expression in PBD005 and PBD018. Our data demonstrate that PEX5 transcripts undergo alternative splicing to produce two mature PEX5 mRNAs. The longer of these but not the shorter is required for the import of PTS2-targeted proteins.

RESULTS

PEX5 transcripts are alternatively spliced

In our initial description of human PEX5 (GenBank accession no. U19721) we noted a second PEX5 cDNA which differed from the first only by a 111 bp insert after position 642 of our published sequence (38). We refer to these two PEX5 cDNAs as PEX5S and PEX5L respectively. We isolated PEX5S and PEX5L cDNAs from skeletal muscle and retinal cDNA libraries and detected both transcripts in RNA from liver, fetal brain and cultured skin fibroblasts by RT-PCR. The 111 bp insert in PEX5L encodes an in-frame 37 amino acid sequence. Amplification and sequencing of genomic DNA from PBD005, PBD018 and 15 unrelated controls showed that the insert sequence is co-linear with the genomic sequence and is flanked by consensus splice donor and acceptor sites (5[prime]-tgtgccccagTTCCT...AGCAGgtaggacatt-3[prime]). These observations indicate that the cDNA insert is an exon which is alternatively spliced to produce the PEX5S and PEX5L transcripts. Our preliminary data on the genomic structure of PEX5 indicates that there are 15 exons; the alternatively spliced exon is exon 8 (N. Braverman, unpublished data).

To confirm expression of the PEX5L transcript by an alternative technique, we hybridized a northern blot of total fibroblast RNA (Fig. 1a, first two lanes) first with a probe corresponding to exon 8 and then with a probe corresponding to PEX5S cDNA. Both probes hybridized to apparently identical 3.3 kb transcripts, confirming that PEX5 transcripts containing exon 8 are expressed. The resolution of the northern blot in this size range was inadequate to resolve transcripts differing by 111 bp. To assess relative abundance of the two PEX5 transcripts in vivo, we compared the intensities of the two fragments amplified from fibroblast cDNA to the fragments amplified from mixtures of known proportions of PEX5L and PEX5S cDNA (Fig. 2). The results suggest that the PEX5L and PEX5S transcripts are expressed in roughly equivalent amounts in these cells. We also identified both transcripts in murine fibroblasts (not shown).


Figure 1. (a) Northern blot analysis of total fibroblast RNA from controls and patients using the alternatively spliced PEX5 exon 8 or PEX5S as probes.(Top) PEX5 exon 8 hybridizes to a transcript of ~3.3 kb in the controls and PBD018, while no corresponding transcript is detected in PBD005. There is non-specific hybridization to a larger 4.4 kb band, corresponding to the 28S rRNA subunit. (Middle) PEX5S cDNA hybridizes to an identical 3.3 kb transcript in controls and PBD018; no hybridizing transcript is detected in PBD005. Similar results were obtained with PEX5L cDNA as probe (not shown). (Bottom) The same blot probed with an OAT cDNA shows a similar amount of RNA in all lanes. (b) Immunoblot analysis of Pex5p in control and patient fibroblasts. Fibroblast protein (15 mg) was subjected to SDS-PAGE, blotted and detected with anti-Pex5p antiserum (1:2000 dilution). This antiserum, directed against the C-terminal two thirds of Pex5p downstream of the region encoded by exon 8, detects both isoforms. Bands corresponding to Pex5p are present in the control and PBD018 samples, but not in that from PBD005. Wild-type Pex5p migrates with an apparent molecular mass of 80 kDa. The faint band at ~65 kDa in PBD018 and PBD005 is a non-specific band detectable in all transformed cell lines (see Materials and Methods), including those transfected with vector alone (not shown).


Figure 2. Relative abundance of PEX5L and PEX5S transcripts in cultured fibroblasts. Fibroblast PEX5 cDNA from two controls (C1 and C2) was amplified with primers flanking the segment encoded by exon 8. To provide a standard, we mixed plasmids containing PEX5L and PEX5S in the indicated proportions and amplified 10 ng of the mix concurrently with the fibroblast cDNA using the same primer pair. The PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. The identity of the amplified fragments was confirmed by sequencing; the 584 bp fragment contains exon 8.

Alignment of Pex5p orthologs

We aligned the sequence of Pex5Lp with orthologs from mouse, Caenorhabditis elegans and yeast (Fig. 3). The overall identity between Pex5Lp and its yeast orthologs is 25-32%, between Pex5Lp and its putative C.elegans ortholog is 36%, and between Pex5Lp and its mouse ortholog is 92%. Interestingly, the region of least sequence similarity between the mammalian and yeast proteins is the middle third, which includes the region encoded by exon 8. Using the PROSITE, BLOCKS and BLASTP programs, we found no known protein motifs in the sequence encoded by exon 8.


Figure 3. Alignment of human Pex5Lp with orthologs from other species. We used LaserGene (DNA STAR, Madison, WI) to perform the alignment. Amino acids identical to the human sequence in at least two other sequences are on a black background. The 37 amino acids encoded by the alternative exon are overlined with a thick bar. TPR repeats are labeled and overlined with a thin bar. Hs, Homo sapiens; Mm, Mus musculus; Ce, Caenorhabditis elegans; Pp, Pichia pastoris; Hp, Hansenula polymorpha; Yl, Yarrowia lipolytica; Sc, Saccharomyces cerevisiase. Throughout the text we number the amino acid sequence relative to Pex5Sp. In PEX5L, the N489K mutation is at residue 526 and the R390ter mutation is at residue 427.

PEX5 expression in patient cell lines

As an initial step to account for the different peroxisomal protein import defects in fibroblasts from the two CG2 patients, we examined their PEX5 mRNA and protein phenotypes. Northern blot analysis using PEX5 exon 8 or the entire PEX5S cDNA as probes did not detect PEX5 transcripts in PBD005 cells (homozygous for the R390ter allele) and detected normal amounts of PEX5 transcripts in PBD018 cells (homozygous for the N489K allele) (Fig. 1a). We were able, however, to detect both short and long PEX5 transcripts in equivalent proportions by RT-PCR of PBD005 fibroblast RNA, indicating that low levels of the mutant transcripts are present in these cells (not shown). Immunoblot analysis of whole fibroblast extracts using a polyclonal antibody directed against the C-terminal two thirds of Pex5p did not detect Pex5p in PBD005 cells and detected an amount about half that of the control in PBD018 cells (Fig. 1b). The observation that the nonsense allele, R390ter, results in reduced levels of transcript and protein is similar to results reported for nonsense mutations in several other genes (67-69).

PEX5L restores both PTS1 and PTS2 import in PBD005 cells

Previously we showed that PBD005 cells are defective in both PTS1 and PTS2 protein import and that transfection with PEX5S cDNA restores PTS1 import and normal peroxisome morphology (7,38). To determine if the PTS2 import defect in PBD005 cells could be corrected by expression of PEX5, we transfected these cells with either pPEX5S or pPEX5L (Fig. 4) and examined them by indirect immunofluorescence microscopy using anti-SKL and anti-thiolase antisera to detect PTS1 and PTS2 protein import respectively. We determined transfection efficiency on a portion of the transfected cell population using anti-Pex5p antiserum and report the data as the number of positive cells divided by the total number of cells counted. Following transfection of PBD005 cells with PEX5S, we found restoration of PTS1 import in 30% of cells (360/1200) (Fig. 4a). In contrast, only 0.3% of cells (5/1687) showed restoration of PTS2 import (Fig. 4b). The transfection efficiency in these experiments was 29% (448/1554). Thus, expression of PEX5S in PBD005 cells restores PTS1 import but has little effect on PTS2 import. In contrast, transfection of these cells with PEX5L restored PTS1 import in 16% of cells (158/1000) and PTS2 import in 14% of cells (355/2518) (Fig. 4c and d respectively). The transfection efficiency for PEX5L in these experiments was 15% (419/2817). Thus, PEX5L expression restores both PTS1- and PTS2-mediated import in virtually all transfected cells and has an ~100-fold greater ability to rescue PTS2 import than does PEX5S.


Figure 4. Expression of PEX5 cDNAs in PBD005 cells. PBD005 fibroblasts were transfected with pPEX5S (a and b) or pPEX5L (c and d). The transfected cell population was permeabilized with Triton X-100 and processed for indirect immunofluorescence using anti-SKL (a and c) or anti-thiolase antiserum (b and d) to detect PTS1 and PTS2 import respectively. Expression of PEX5S cDNA restores PTS1, but not PTS2 import [contrast the punctate staining in (a) with the cytoplasmic staining in (b)]. Expression of PEX5L cDNA restores both PTS1 and PTS2 import [note the punctate staining in both (c) and (d)]. No punctate staining was observed in transfections with vector alone (not shown). The intraperoxisomal location of thiolase and PTS2-CAcT (see below) was confirmed in this and subsequent experiments (Figs 5 and 6) by demonstrating the absence of punctate immunofluoresce in digitonin-permeabilized cells (see Materials and Methods).

We also assessed the effect of expression of PEX5L on the import of a heterologous PTS2-targeted protein. In these experiments, we utilized pPTS2-CAcT, a construct which encodes a chimeric protein with the N-terminal PTS2 peptide of rat thiolase added in-frame to the N-terminus of bacterial chloramphenicol acetyltransferase (CAcT) (13). When expressed in mammalian cells, unmodified CAcT is localized to the cytosol. In cells with an intact PTS2 import pathway, addition of the N-terminal PTS2 targeting sequence directs CAcT to peroxisomes (6,13,31,33). We co-transfected PBD005 cells with pPTS2-CAcT and either pPEX5S or pPEX5L (Fig. 5). To determine PTS2 and PTS1 import, we stained the transfected cell population with anti-CAcT and anti-SKL antibodies respectively and determined the number of PTS2-CAcT-positive cells with a peroxisomal (punctate) pattern divided by the total number of PTS2-CAcT-expressing cells. To limit our analysis to those cells in which there was co-expression of pPEX5 and pPTS2-CAcT, we only counted cells with a punctate PTS1 pattern as detected with the anti-SKL antibody. In PBD005 cells co-transfected with pPTS2-CAcT plus pPEX5S, we observed no instances of normal PTS2-mediated import. PTS2-CAcT was limited to the cytosol in nearly all transfected cells (Fig. 5a). Occasionally (29/446 or 6.5% of all PTS2-CAcT-positive cells), we observed cells with a few immunofluorescent punctate structures which co-localized with peroxisomal markers. These punctate structures numbered [le]20/cell, were always present in cells with a background of cytosolic PTS2-CAcT and represented a small fraction of the total peroxisomes present in the cell as assessed by PTS1 staining (Fig. 5c and d). In contrast, in PBD005 cells co-transfected with pPTS2-CAcT plus pPEX5L, 100% of the PTS2-CAcT-positive cells (576/576) imported the marker protein into punctate structures which co-localized with peroxisomes (Fig. 5e and f). In all positive cells there was import into nearly all recognizable peroxisomes with little or no residual cytosolic PTS2-CAcT. Thus, when expressed in PBD005 cells, Pex5Lp efficiently rescues the peroxisomal import of PTS2-CAcT while Pex5Sp does not.


Figure 5. Co-expression of PEX5 cDNAs and a PTS2 marker protein in PBD005 cells. PBD005 cells were co-transfected with pPTS2-CAcT and either pPEX5S or pPEX5L. The transfected cell population was stained with anti-CAcT monoclonal antibody, to detect PTS2 import, and anti-SKL, to detect PTS1-mediated import into peroxisomes. In nearly all cells expressing pPEX5S, PTS2-CAcT remains cytosolic (a and b). In a few transfected cells (6.5%), a few punctate structures which co-localize with peroxisomes are present (c, arrows, and d). In contrast, in cells expressing PEX5L, PTS2-CAcT is exclusively peroxisomal (e and f). No punctate structures were present after transfection with pPTS2-CAcT alone (not shown).

pPEX5L-N489K restores PTS2 import in PBD005 cells

Since the only difference between PEX5L and PEX5S is the presence of the additional exon (exon 8) in PEX5L, we suggest that the region of the protein encoded by this sequence is required for the effect of PEX5L on PTS2 import. Preservation of PTS2 import in PBD018 cells would be explained by the normal amount of Pex5Lp, regardless of the missense mutation, N489K, 275 residues downstream of the insert. To test this hypothesis, we co-transfected PBD005 cells with pPTS2-CAcT and either pPEX5L-N489K or pPEX5S-N489K. Subsequently, we stained the transfected cell population with an anti-CAcT antibody to detect PTS2 protein import and an anti-PMP70 antibody to mark peroxisomes (Fig. 6). We found that 100% of the PTS2-CAcT-positive cells (200/200) exhibited a punctate pattern of immunofluorescence (Fig. 6a) which co-localized with peroxisomes (Fig. 6b). In contrast, co-transfection of PBD005 cells with pPEX5S-N489K and pPTS2-CAcT did not restore PTS2 protein import (no punctate cells were seen in 212 counted) (Fig. 6e). As expected, neither the short nor long isoform of PEX5-N489K rescued PTS1 import (not shown). We conclude that the N489K mutation, which alters the sixth TPR domain, does not affect the function Pex5p plays in PTS2 import. These results are consistent with the preservation of PTS2 import previously observed in PBD018 cells (6,7,38,39).


Figure 6. Co-expression of mutant PEX5 cDNAs and pPTS2-CAcT in PBD005 cells. PBD005 cells were co-transfected with pPTS2-CAcT and either pPEX5L-N489K (a and b) or pPEX5L-R390ter (c and d). The transfected cell population was stained with an anti-CAcT monoclonal antibody, to detect PTS2 import, and an anti-PMP70 antiserum, to detect peroxisomes. Expression of both mutant proteins restores PTS2 import, as seen by the punctate pattern of immunofluorescence detected with anti-CAcT antibodies (a and c), which co-localizes with peroxisomes detected by anti-PMP70 antiserum (b and d). For PBD005 cells co-transfected with pPTS2-CAcT and either pPEX5S-N489K (e) or pPEX5S-R390ter (f), the location of PTS2-CAT is cytosolic.

pPEX5L-R390ter restores PTS2 import in PBD005 cells

The most likely explanation for the PTS2 import defect in PBD005 cells is the virtual absence of PEX5 transcripts and, consequently, protein in these cells (Fig. 1). Alternatively, it is formally possible that low level expression of the Pex5p-R390ter protein interferes with PTS2-mediated import in PBD005 cells. To distinguish between these possibilities, we co-transfected PBD005 cells with pPEX5L-R390ter and pPTS2-CAcT. Our results were unequivocal; expression of Pex5Lp-R390ter restored PTS2 import, as is evident from the punctate pattern of PTS2-CAcT in 100% of cells (824/824) co-localizing with peroxisomes (Fig. 6c and d). In contrast, we observed no punctate staining in PBD005 cells transfected with pPEX5S-R390ter (no punctate cells in 200 counted) (Fig. 6f). We confirmed that PBD005 cells transfected with pPEX5L-R390ter express substantial amounts of the truncated Pex5Lp by immunoblot analysis. Pex5Lp-R390ter (apparent molecular mass 60 kDa, as compared with 80 kDa for wild-type Pex5p) was present in amounts greater than Pex5p in control cells and there was no evidence of the translational apparatus reading through the nonsense mutation (not shown). Neither the short nor long isoforms of Pex5p-R390ter rescued PTS1 import (not shown). These results show that the PTS2 import defect in PBD005 cells results from the virtual absence of PEX5L mRNA and protein and are also in agreement with our hypothesis that the region of Pex5p encoded by exon 8 is necessary for PTS2 import.

DISCUSSION

Pex5p is required for both PTS1 and PTS2 protein import

The identification of human PEX genes and characterization of the cellular phenotypes of PBD patients are initial steps in understanding mammalian peroxisome biogenesis. Previously, we and others have shown that mutations in PEX5, the gene encoding the PTS1 receptor, account for CG2 of the PBD (38,39). Surprisingly, the cellular phenotype of one CG2 patient included deficiency of both PTS1- and PTS2-mediated import. This combined import defect was not predicted by studies of yeast PEX5 mutants, which are defective only in PTS1-mediated import (59-63).

Here we show that the human PEX5 transcript undergoes alternative splicing to produce two mature transcripts (PEX5S and PEX5L) differing only by the presence of a 111 bp internal exon, which our genomic studies have tentatively identified as exon 8 of the PEX5 structural gene. RT-PCR amplification of mRNA indicates that PEX5S and PEX5L are present in roughly equivalent amounts in cultured fibroblasts. Alignment of human Pex5Lp with orthologs from mouse and yeast shows that the portion of the sequence encoded by exon 8 is not well conserved between mammals and yeast, supporting a novel function for PEX5L. Our experiments show that the PEX5L isoform is required for the import of PTS2 proteins, thus providing a mechanism for convergence of the PTS1 and PTS2 import pathways in mammals. Consistent with our observations, Tsukamoto et al. (66) and Otera et al. (65) recently described mutant CHO cell clones with primary defects in PEX5. Like the PBD005 cells, certain of these clones are defective in both PTS1- and PTS2-mediated import. Furthermore, Baes et al. (70) report that mice homozygous for a targeted disruption of the PEX5 gene were also defective in both matrix protein import pathways.

Human PEX5 cDNAs have also been described by two other groups. Fransen et al. (40) isolated a cDNA identical to PEX5L (GenBank accession no. X84899) from a liver cDNA library but did not express this cDNA in PBD patient cell lines. Wiemer et al. (39) isolated a cDNA identical to PEX5S (GenBank accession no. Z48054) and showed that expression of this cDNA in PBD005 cells (referred to in their paper as FAIR-T) restored PTS2 protein import in `only some but not all' transfected cells. In contrast with Wiemer et al. (39), we found virtually undetectable (<1%) restoration of PTS2 import in PEX5S-transfected cells stained for an endogenous PTS2-targeted protein, peroxisomal thiolase. Similarly, in co-transfection experiments with PEX5S and the heterologous protein PTS2-CAcT, we found no recovery of normal PTS2 import and a low level of incomplete rescue (6.5% of expressing cells). Incomplete rescue was characterized by [le]20 PTS2-CAcT-containing peroxisomes per cell and with persistence of PTS2-CAcT in the cytosol. In contrast, the recovery of PTS2 import in cells transfected with PEX5L, as measured by localization of endogenous thiolase or heterologous PTS2-CAcT, was dramatic and occurred in essentially 100% of transfected cells. This pronounced difference in recovery of PTS2 import following expression of PEX5L as compared with PEX5S suggests that it is primarily, if not exclusively, the long isoform of PEX5 which is required for PTS2 import.

The explanation for the low but discernible level of PTS2 import following transfection of PBD005 cells with PEX5S is uncertain. The observation of occasional cells with some punctate peroxisomal staining following transfection with PEX5S was reproducible and clearly different from untransfected cells (7; this paper) or cells transfected with vector alone (39; this paper), which show no punctate staining. Thus, this low level of punctate staining is unlikely to represent either residual PTS2 uptake independent of Pex5Lp or a few cells with adequate amounts of mutant (truncated) Pex5Lp. Perhaps a small amount of truncated Pex5Lp present in a few PBD005 cells is stabilized or augmented in some way by the high levels of Pex5Sp introduced by transfection. This possibility is supported by our detection of some PEX5L transcript in PBD005 cells using sensitive RT-PCR methods.

What is the function of Pex5Lp in PTS2 protein import?

At least two models for the role of Pex5Lp in PTS2 import are consistent with our results. The simplest model has Pex5Lp function as a receptor for both PTS1 and PTS2 proteins. However, this dual ligand receptor model is untenable given the recent identification of a discrete PTS2 receptor, Pex7p, defective in PBD CG11 (RCDP). Cells from RCDP patients homozygous for a PEX7 nonsense mutation have a complete defect of PTS2 import, indicating that there is no alternative import pathway for these proteins independent of Pex7p (31-33). Additionally, RCDP cells transfected with PEX5L expression constructs do not recover PTS2 import (not shown).

In an alternative, and in our view more likely, model Pex5Lp is somehow required for normal function of Pex7p, the PTS2 receptor. This receptor interaction model would depend on the portion of PEX5L encoded by exon 8 and would not require import (either prior or simultaneous) of PTS1 proteins. PBD018 cells import PTS2-targeted proteins normally despite their inability to import PTS1 proteins and expression of Pex5Lp-R390ter or Pex5Lp-N489K rescues PTS2, but not PTS1, import in PBD005 cells. Furthermore, it is unlikely that this proposed interaction requires the C-terminal Pex5Lp TPR domain, since mutant receptors with a truncation or amino acid substitution in this region (Pex5Lp-R390ter and Pex5Lp-N489K respectively) restore PTS2 import. We favor the idea that the interaction involves association of Pex5Lp with a Pex7p-PTS2 protein complex in the cytosol with subsequent transport of the entire complex to the peroxisome membrane. Zhang et al. (56) suggest that the N-terminus of yeast Pex7p may function as a novel PTS. Our model would explain their result if the N-terminus of Pex7p interacts with Pex5Lp rather than the peroxisome. Associations between TPR proteins (Pex5p) and WD40 proteins (Pex7p) have been reported in other systems (41,54). Although yeast Pex5p and Pex7p interact only indirectly in a two hybrid assay, possibly requiring a third protein such as Pex14p (W. Kunau, personal communication), mammalian Pex5Lp and Pex7p do interact in this system (S. Gould and G. Dodt, unpublished data). Our results indicate that this interaction is required for PTS2 import in mammalian cells and provides a potential mechanism for coordination of the metabolic capabilities of the peroxisome in different tissues and in response to various physiological stimuli. Interestingly, our receptor interaction model predicts that certain PEX5 mutations may selectively inactivate its ability to interact with Pex7p without altering its PTS1 receptor function. Such mutations should cause a phenotype similar to RCDP.

The importance of alternative splicing of PEX5 is reflected in its conservation in other mammalian species. We observed alternative splicing of murine PEX5 exon 8 (data not shown) and very recently Otera et al. (65) described the occurrence of similar alternative splicing of the CHO PEX5 transcript, although they did not determine the relative abundance of the two transcripts. These investigators identified a mutant CHO clone with a PEX5 missense mutation in the first TPR domain and defective import of both PTS1- and PTS2-targeted proteins. Unfortunately, they did not examine the consequence of this missense mutation on the stability of Pex5p. Our results predict that it may have destabilized the protein. In agreement with our data, Otera et al. showed that expression of the long but not the short isoform of Pex5p restored PTS2-mediated import in these cells and also suggested a model involving interaction of Pex5Lp with Pex7p (65).

A molecular explanation for the different patient phenotypes

Our results provide an explanation for the differences in the clinical and biochemical phenotypes of the two CG2 patients. PBD005 had ZS, the most severe PBD phenotype, and a profound defect in plasmalogen biosynthesis (38). In fact, the plasmalogen levels in PBD005 cells are reduced to an extent equivalent to those in RCDP cells, which are the lowest of any PBD patients (2). DHAP-AS, which catalyzes an initial step in the plasmalogen biosynthesis pathway, is a PTS2-targeted peroxisomal protein (18). Thus, the severe defect in plasmalogen biosynthesis exhibited by PBD005 cells is consistent with the complete loss of PTS2 import due to the virtual absence of Pex5Lp.

MATERIALS AND METHODS

Cell lines and transfection studies

Patient cell lines are assigned a unique PBD number used in all publications from our group. Primary skin fibroblasts from PBD005 and PBD018 were transformed as described (38). We transfected these cells with lipofectAMINE[trade] (Gibco BRL) according to the manufacturer's suggestions or by electroporation as described (31) and performed indirect immunofluorescence 48 h later. We repeated all transfection experiments at least three times. The variance of the mean percent recovery of peroxisome import for individual experiments was <20% of the reported average mean calculated from all experiments.

Molecular studies of PEX5

We cloned PEX5 cDNAs from multiple tissues as reported (38). To determine the intronic sequence flanking exon 8, we amplified genomic DNA using primer pair DV1239 (sense, 5[prime]-GAGGATCTGCAGCACACG-3[prime], exon 7) and DV997 (antisense, 5[prime]-GTCTTGTGAACTGGTCAACC-3[prime], exon 9). For the data in Figure 2, we used primer pair DV1038 (sense, 5[prime]-CTGTGGATGTAACTCAGG-3[prime]) and DV946 (antisense, 5[prime]-CTTATCATAGGTAGCTGACG-3[prime]). Sequencing was performed with Sequenase v.2.0 (US Biochemical). Unless otherwise stated, PCR reactions contained 100 ng template DNA, 10 pmol each primer, 100 µM dNTPs, 2.5 U Taq DNA polymerase and 1× buffer (1.5 mM MgCl2; Boehringer Mannheim) in 50 µl. Initial denaturation was 95°C for 6 min followed by 30 cycles of annealing (57-62°C), extension (72°C) and denaturation (95°C) for 1 min each, with a 10 min final extension. We reverse transcribed total fibroblast RNA using Superscript reverse transcriptase (Gibco BRL) and the gene-specific primer DV1041 (antisense, 5[prime]-CTCACAGGG- AATCAGAGC-3[prime], complementary to sequence in the 3[prime]-UTR) according to the manufacturer's protocol.

Plasmids

pPEX5S contains the complete PEX5S ORF in pcDNA3 (Invitrogen). We made pPEX5S-N489K by replacing the HindIII-XhoI fragment from pPEX5S with the corresponding fragment of a pPEX5 RT-PCR clone from PBD018 cells. Similarly, we made pPEX5S-R390ter using the HindIII-XhoI fragment (containing R390ter) of a pPEX5 RT-PCR clone from PBD005 cells. To produce control and mutant PEX5L constructs, we replaced the Sse8387-HincII fragment of pPEX5S, pPEX5S-N489K and pPEX5S-R390ter with the corresponding fragment from a control pPEX5L RT-PCR clone (containing exon 8). To amplify the Sse8387-HincII fragment we used primer pair DV1037 (sense, 5[prime]-TCCTGCAGGACCAGAATGC-3[prime]) and DV946 (antisense, 5[prime]-CTTATCATAGGTAGCTGACG-3[prime]). To amplify the HindIII-XhoI fragment we used DV947 (sense, 5[prime]-GTTGACCAGTTCACAAGACC-3[prime]) and DV1054 (antisense, 5[prime]-CAGAAAGTGCTCCACAGC-3[prime]). We cloned the amplified cDNA products into pCR[trade] using a TA Cloning Kit (Invitrogen). pPTS2-CAcT cDNA encodes a chimeric protein with the N-terminal PTS2 signal of rat thiolase followed by bacterial CAcT (13). We verified all constructs by sequencing the complete insert.

Antibodies and indirect immunofluorescence

We produced an anti-Pex5p antiserum by immunizing rabbits with a fusion protein comprised of maltose binding protein fused to the C-terminal two thirds of Pex5Sp (residues 220-602) (38). This antiserum detects both Pex5p isoforms, as well as the mutant proteins produced from the two patient alleles. Rabbit anti-thiolase antiserum was a gift from R. Rachubinski. Rabbit anti-SKL antiserum, which recognizes multiple PTS1-containing proteins (71), and anti-PMP70 antiserum (72) have been described. We purchased monoclonal anti-CAcT and anti-myc antibodies (Berkeley Antibody, Richmond, CA) and all secondary antibodies (Kirkgaard and Perry Laboratories, Gaithersberg, MD).

Our procedure for indirect immunofluorescence has been reported (73). Briefly, we fix and permeabilize cells grown on coverslips by incubating them in 1% Triton X-100 for 5 min. To discriminate between antigens located inside the peroxisome and those on the cytosolic surface of the peroxisome membrane, we incubate cells with digitonin (25 µg/ml for 5 min), which permeabilizes the plasma membrane but leaves the peroxisomal membrane intact so that intraperoxisomal antigens are not detected (13,74). All micrographs were with a Zeiss Axiophot microscope using Kodak Ektachrome ASA 400 film. There was no `bleed-through' fluorescence when the FITC or rhodamine/Texas Red-labeled cells were viewed through the red or green filter respectively (not shown).

Characterization of patient cell lines

We isolated total fibroblast RNA with either RNAzol[trade] B (Cinna/Biotecx, Friendswood, TX) or guanidinium thiocyanate (67). For northern blot analysis, total cellular RNA (10 µg/lane) was electrophoresed and transferred as described (75). The blot was probed sequentially with PEX5 exon 8, full-length PEX5S and PEX5L cDNA. The exon 8 probe was a 110 bp fragment generated by amplification of pPEX5L using primers DV1320 (sense, 5[prime]-TTCCTGAAA TTCGTGCGG-3[prime]) and DV1321 (antisense, 5[prime]-TGCTGCTGTATACTCTGC-3[prime]), located within exon 8. We synthesized radiolabeled probes using specific primers (exon 8) or random hexamers (PEX5S and PEX5L) as described (76). The blot probed with exon 8 was washed with 2× SSC, 0.1% SDS at 50°C, all others were washed with 0.1× SSC, 0.1% SDS at 50°C. The procedures for SDS-PAGE and immunoblotting have been described (67).

Sequence alignment and protein structure analysis

We performed multiple sequence alignments using LaserGene (DNA STAR, Madison, WI) with modification by visual inspection. To calculate percent identity, we divided the number of identical amino acids by the length of the shorter complete sequence (77). The GenBank accession number for the putative C.elegans Pex5p ortholog is Z66494.

ACKNOWLEDGEMENTS

This work was supported in part by a NIH grant to the Kennedy Krieger Institute (PO1HD10981; S.G. and D.V.) and to the General Clinical Research Centers (RR00052 and RR00722; N.B.). We thank Ann and Hugo Moser for their continued support of this work and Sandy Muscelli for assistance with preparation of this manuscript. D.V. is an Investigator in The Howard Hughes Medical Institute.

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*To whom correspondence should be addressed at: PCTB 802, Johns Hopkins University, 725 North Wolfe Street, Baltimore, MD 21205, USA. Tel: +1 410 955 4260; Fax: +1 410 955 7397; Email: david.valle@qmail.bs.jhu.edu

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