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.     

Characterization of a cDNA encoding cottonseed catalase

A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.     

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.     

The C-terminal domain of plant catalases. Implications for a glyoxysomal targeting sequence.

A castor bean (Ricinus communis cv. Hale) cDNA encoding catalase was cloned and sequenced. The cDNA encoding the carboxy-terminal domain of catalase was compared to the corresponding sequences of six other plant catalases. The deduced amino acid sequences were compared according to the chemical attributes of each amino acid within each carboxy-terminal domain. A tripeptide sequence having the chemical attributes of the peroxisomal targeting sequence [Gould, S.J., Keller, G.-A., Hosken, N., Wilkinson, J. & Subramani, S. (1989) J. Cell Biol. 108, 1657-1664] was common to all the glyoxysomal/peroxisomal plant catalases. This sequence motif was located six amino acids from the carboxy terminus of each of the plant catalases. An identical motif was also found within the carboxy-terminal domain of three mammalian catalases previously sequenced. We hypothesize that these motifs are at least part of the targeting mechanism for catalase entry into plant glyoxysomes/peroxisomes.     

Two genes encode the two subunits of cottonseed catalase

The isolation and sequence of a cDNA encoding a developmentally distinct subunit of cottonseed catalase are presented. A 1.8-kb cDNA was selected from a cDNA library constructed with poly(A)+ RNA isolated from 3-day-old dark-grown cotyledons in which a second subunit (designated SU 2 in an earlier publication) of catalase was predominantly synthesized. The cDNA encodes a 492-amino acid peptide with a calculated Mr of 56,900. The nucleotide sequence is 76% identical to a cDNA encoding another subunit (SU 1) which was predominantly synthesized in 1-day-old-cotyledons. Most of the divergence occurs in the 5' and 3' non-coding regions, and at the third positions of the codons. The deduced amino acid sequence is 92% identical to that of SU 1. Denaturing isoelectric focusing and SDS-PAGE of products transcribed and translated in vitro from these cDNAs revealed that the cDNA selected from the "1-day" library encoded SU 1 and the cDNA selected from the "3-day" library (this paper) encoded SU 2 of catalase. These data and results from Southern blot analyses of genomic DNA indicate that there are two genes encoding catalase subunits in cotton cotyledons, with only one copy of SU 1 and at least two copies of SU 2 in the genome. A peroxisomal targeting signal, e.g., Ser-Lys-Leu, is not located at the C-terminus of either subunit, or within 25 residues of the C-terminus of SU 1, although it occurs at six residues upstream from the C-terminus of SU 2. A possible location of a targeting sequence for catalase and other peroxisomal proteins lacking the C-terminal tripeptide motif is proposed.     

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

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

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

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

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

A conserved tripeptide sorts proteins to peroxisomes

The firefly luciferase protein contains a peroxisomal targeting signal at its extreme COOH terminus (Gould et al., 1987). Site-directed mutagenesis of the luciferase gene reveals that this peroxisomal targeting signal consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine. When this tripeptide is appended to the COOH terminus of a cytosolic protein (chloramphenicol acetyltransferase), it is sufficient to direct the fusion protein into peroxisomes. Additional mutagenesis experiments reveal that only a limited number of conservative changes can be made in this tripeptide targeting signal without abolishing its activity. These results indicate that peroxisomal protein import, unlike other types of transmembrane translocation, is dependent upon a conserved amino acid sequence.     

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.     

Temperature-sensitive phenotype of Chinese hamster ovary cells defective in PEX5 gene

SK32 mutant cells, which were isolated as peroxisome-deficient Chinese hamster ovary (CHO) cells by an advantage of a visible peroxisome form of green fluorescent protein (GFP), were found to suffer from a functional loss of PEX5 gene encoding for PTS1R. The sequence analysis of cDNA indicated that PEX5 gene encoded for the two isoforms composed of 603 amino acids (PTS1RS) and 640 amino acids (PTS1RL). The mutation changed glycine to arginine at amino acid position 343 of PTS1RL (corresponding to the position 306 of PTS1RS) in SK32 cells. The mutant cells exhibited a temperature-sensitive (TS) phenotype on the peroxisomal localizations of the recombinant GFP and urate oxidase appending a genuine peroxisome targeting signal 1 (PTS1), a tripeptide of Ser-Lys-Leu (SKL) at the C-terminus, but did not on that of catalase harboring a divergent PTS1, Lys-Ala-Asn-Leu (KANL) sequence. 3-ketoacyl-CoA thiolase (hereafter referred to as thiolase), which harbors an extension sequence (PTS2) at the N-terminus, never appeared to be affected on the peroxisomal localization in the mutant cells. When thiolase was examined on the molecular size in the mutant cells, the enzyme existed as the larger precursor form in the peroxisomes at 37 degrees C and a considerable part (almost half) was converted to the mature size at 30 degrees C. These results indicate that the amino acid substitution, Gly306Arg in PTS1RS and/or Gly343Arg in PTSRL, gives rise to TS phenotype on the peroxisomal translocation of PTS1 proteins and the maturation of PTS2 protein.     

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.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Identification of the peroxisomal targeting signal for cottonseed catalase

Catalase is a ubiquitous peroxisomal matrix enzyme, yet the molecular targeting signal(s) for sorting it in plant cells has not been defined. The most common peroxisome targeting signal (PTS) is a C-terminal tripeptide composed of a conserved SKL motif (type 1 PTS). The PTS for cottonseed catalase (Ccat) was elucidated in this study from immunofluorescence microscopic analyses of tobacco BY-2 suspension cells serving as an in vivo import system. To distinguish biolistically introduced Ccat from endogenous tobacco catalase, Ccat was hemagglutinin (HA) epitope-tagged at its N-terminus. Bombardment with HA-Ccat resulted in the import of Ccat into glyoxysomes, the specialized type of peroxisome in BY-2 cells. The C-terminal tripeptide of Ccat, PSI, is necessary for import. Evidence for this were mislocalizations to the cytosol of PSI-truncated Ccat and AGV-substituted (for PSI) Ccat. PSI-COOH, however, was not sufficient to re-route chloramphenicol acetyltransferase (CAT) from the cytosol to glyoxysomes, whereas the Ccat tetrapeptide RPSI-COOH was sufficient. Surprisingly, substitution of K (common at the fourth position in other plant catalases) for the R (CAT-KPSI) decreased import efficiency. However, substitution of K did not affect import, when additional upstream residues in Ccat were included (e.g. CAT-NVKPSI). Other evidence for the importance of upstream residues comprised abolishment of Ccat import due to substitutions with non-conserved residues (e.g. -AGVNVRPSI for -SRLNVRPSI). These data indicate that Ccat is sorted to plant peroxisomes by a degenerate type 1 PTS (PSI-COOH) whose residues are functionally dependent on a strict context of adjacent C-terminal amino acid residues.     

Substrate specificity of the isoprenylated protein endoprotease.

Proteins containing a CAAX motif at their carboxyl termini are subject to isoprenylation at the cysteine residue. Proteolytic trimming of isoprenylated proteins is essential in the activation of these proteins. A microsomal endopeptidase activity has been identified which cleaves all-trans farnesylated cysteine containing tetrapeptides between the modified residue and the adjacent amino acid to liberate the modified cysteine residue and an intact tripeptide. Structure/activity studies are reported here on this endopeptidase activity which are consistent with the premise that this protease is identical to the one normally involved in the cellular isoprenylation pathway. The protease only processes peptides which possess an isoprenyl moiety. Within the isoprenyl series, the enzyme hydrolyzes all-trans-farnesyl-, all-trans-geranylgeranyl-, and geranyl-containing peptides. The protease also recognizes the AAX sequence, because the protease behaves either stereospecifically or stereoselectively with respect to the individual amino acids of the tripeptide. The enzyme only measurably hydrolyzes isoprenylated peptides possessing L-amino acids at C and A. On the other hand, there is a small but measurable hydrolysis of isoprenylated peptides containing a D-amino acid at X.     

Importance of sequences adjacent to the terminal tripeptide in the import of a peroxisomal Candida tropicalis protein in plant peroxisomes

 The peroxisome targeting signal (PTS) required for import of the rat acyl-CoA oxidase (AOX; EC 1.3.3.6) and the Candida tropicalis multifunctional protein (MFP) in plant peroxisomes was assessed in transgenic Arabidopsis thaliana (L.) Heynh. The native rat AOX accumulated in peroxisomes in A. thaliana cotyledons and targeting was dependent on the presence of the C-terminal tripeptide S-K-L. In contrast, the native C. tropicalis MFP, containing the consensus PTS sequence A-K-I was not targeted to plant peroxisomes. Modification of the carboxy terminus to the S-K-L tripeptide also failed to deliver the MFP to peroxisomes while addition of the last 34 amino acids of the Brassica napus isocitrate lyase, containing the terminal tripeptide S-R-M, enabled import of the fusion protein into peroxisomes. These results underline the influence of the amino acids adjacent to the terminal tripeptide of the C. tropicalis MFP on peroxisomal targeting, even in the context of a protein having a consensus PTS sequence S-K-L. Received: 19 July 1999 / Accepted: 19 February 2000     

Non-canonical peroxisome targeting signals: identification of novel PTS1 tripeptides and characterization of enhancer elements by computational permutation analysis

ABSTRACT: BACKGROUND: High-accuracy prediction tools are essential in the post-genomic era to define organellar proteomes in their full complexity. We recently applied a discriminative machine learning approach to predict plant proteins carrying peroxisome targeting signals (PTS) type 1 from genome sequences. For Arabidopsis thaliana 392 gene models were predicted to be peroxisome-targeted. The predictions were extensively tested in vivo, resulting in a high experimental verification rate of Arabidopsis proteins previously not known to be peroxisomal. RESULTS: In this study, we experimentally validated the predictions in greater depth by focusing on the most challenging Arabidopsis proteins with unknown non-canonical PTS1 tripeptides and prediction scores close to the threshold. By in vivo subcellular targeting analysis, three novel PTS1 tripeptides (QRL>, SQM>, and SDL>) and two novel tripeptide residues (Q at position -3 and D at pos. -2) were identified. To understand why, among many Arabidopsis proteins carrying the same C-terminal tripeptides, these proteins were specifically predicted as peroxisomal, the residues upstream of the PTS1 tripeptide were computationally permuted and the changes in prediction scores were analyzed. The newly identified Arabidopsis proteins were found to contain four to five amino acid residues of high predicted targeting enhancing properties at position -4 to -12 in front of the non-canonical PTS1 tripeptide. The identity of the predicted targeting enhancing residues was unexpectedly diverse, comprising besides basic residues also proline, hydroxylated (Ser, Thr), hydrophobic (Ala, Val), and even acidic residues. CONCLUSIONS: Our computational and experimental analyses demonstrate that the plant PTS1 tripeptide motif is more diverse than previously thought, including an increasing number of non-canonical sequences and allowed residues. Specific targeting enhancing elements can be predicted for particular sequences of interest and are far more diverse in amino acid composition and positioning than previously assumed. Machine learning methods become indispensable to predict which specific proteins, among numerous candidate proteins carrying the same non-canonical PTS1 tripeptide, contain sufficient enhancer elements in terms of number, positioning and total strength to cause peroxisome targeting.     

The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals

We describe the cloning of the Hansenula polymorpha PER1 gene and the characterization of the gene and its product, PER1p. The gene was cloned by functional complementation of a per1 mutant of H. polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim- phenotype). The DNA sequence of PER1 predicts that PER1p is a polypeptide of 650 amino acids with no significant sequence similarity to other known proteins. PER1 expression was low but significant in wild-type H. polymorpha growing on glucose and increased during growth on any one of a number of substrates which induce peroxisome proliferation. PER1p contains both a carboxy- (PTS1) and an amino- terminal (PTS2) peroxisomal targeting signal which both were demonstrated to be capable of directing bacterial beta-lactamase to the organelle. In wild-type H. polymorpha PER1p is a protein of low abundance which was demonstrated to be localized in the peroxisomal matrix. Our results suggest that the import of PER1p into peroxisomes is a prerequisite for the import of additional matrix proteins and we suggest a regulatory function of PER1p on peroxisomal protein support.