©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
PER3, a Gene Required for Peroxisome Biogenesis in Pichia pastoris, Encodes a Peroxisomal Membrane Protein Involved in Protein Import (*)

Henry Liu (1), Xuqiu Tan (1), Kimberly A. Russell (1), Marten Veenhuis (2), James M. Cregg (1)(§)

From the (1) Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000 and (2) Laboratory for Electron Microscopy, Biological Center, University of Groningen, 9751 NN Haren, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

PER genes are essential for the biogenesis of peroxisomes in the yeast Pichia pastoris. Here we describe the cloning of PER3 and functional characterization of its product Per3p. The PER3 sequence predicts that Per3p is a 713-amino acid (81-kDa) hydrophobic protein with at least three potential membrane-spanning domains. We show that Per3p is a membrane protein of the peroxisome. Methanol- or oleate-induced cells of per3-1, a mutant strain generated by chemical mutagenesis, lack normal peroxisomes but contain numerous abnormal vesicular structures. The vesicles contain thiolase, a PTS2 protein, but only a small portion of several other peroxisomal enzymes, including heterologously expressed luciferase, a PTS1 protein. These results suggest that the vesicles in per3-1 cells are peroxisomal remnants similar to those observed in cells of patients with the peroxisomal disorder Zellweger syndrome, and that the mutant is deficient in PTS1 but not PTS2 import. In a strain in which most of PER3 was deleted, peroxisomes as well as peroxisomal remnants appeared to be completely absent, and both PTS1- and PTS2-containing enzymes were located in the cytosol. We propose that Per3p is an essential component of the machinery required for import of all peroxisomal matrix proteins and is composed of independent domains involved in the import of specific PTS groups.


INTRODUCTION

Peroxisomes (glyoxysomes, glycosomes) are ubiquitous, eukaryotic organelles that are enclosed by a single membrane and are the site of hydrogen peroxide-generating oxidases as well as catalase, which decomposes this toxic by-product into water and oxygen. An unusual feature of peroxisomes is that the specific pathways that involve the organelles vary greatly, depending upon the organism (1, 2) . Furthermore, their size, number, and enzymatic content within a single organism or tissue can change dramatically in response to environmental stimuli (3, 4) . Peroxisomes have no DNA; therefore, all peroxisomal proteins are likely to be encoded by nuclear DNA (5) . Proteins destined for the peroxisome are synthesized on free polysomes and post-translationally imported into the organelle in an ATP-dependent manner (6, 7, 8) . Two peroxisomal targeting signals (PTSs)() have been characterized in detail (9) . The first, PTS1, is a tripeptide motif, Ser-Lys-Leu (and conservative variants), that is located at the carboxyl terminus of many peroxisomal matrix proteins (10, 11) . Proteins that end in PTS1 are properly imported into peroxisomes of animals and fungi, glyoxysomes of plants, and glycosomes of trypanosomes (12, 13) . Thus, the PTS1 system has been conserved throughout evolution. The second, PTS2, is characterized by the consensus sequence (RL/IXQ/HL) located near the amino terminus of some peroxisomal proteins including: 3-ketoacyl-CoA thiolases from rats, humans, and several yeast species; watermelon malate dehydrogenase; and amine oxidase and the peroxisome biogenesis protein Per1p from the yeast Hansenula polymorpha(14, 15, 16, 17, 18, 19, 20) . Evidence suggests that additional PTSs exist internally within some peroxisomal proteins including: Candida tropicalis acyl-CoA oxidase (21, 22) , Saccharomyces cerevisiae catalase (23) , Candida boidinii 47-kDa peroxisomal membrane protein PMP47 (24) , and H. polymorpha malate synthase (25) .

In humans, peroxisomes are indispensable for survival as demonstrated by a family of lethal genetic disorders collectively referred to as Zellweger syndrome in which normal peroxisomes appear to be absent (26) . In cell lines derived from these patients, peroxisomal enzymes remain in the cytosol and are either present and active, or absent due to rapid degradation (27, 28) . Cell fusion studies with Zellweger cell lines indicate that the disease is the consequence of defects in any one of at least nine different genes (29, 30) . Recently human cDNA clones were identified that restored normal organelles to two different Zellweger complementation groups (29, 31) . Although normal peroxisomes are absent from or grossly deficient in Zellweger cells, abnormal vesicles are observed (32, 33) . These vesicles contain peroxisomal membrane proteins, and thus are believed to be peroxisomal remnants, termed ghosts. Furthermore, the ghost structures in some Zellweger lines contain the PTS2 enzyme thiolase, suggesting that these lines have a functional PTS2 import system and must be defective in one or more other import systems (34, 35) .

We have selected the methylotrophic yeasts Pichia pastoris and H. polymorpha as model systems to study the molecular mechanisms involved in the biogenesis of peroxisomes. In P. pastoris, peroxisome-deficient ( per) mutants have been isolated that are affected in 10 different genes ( PER genes) (36, 37) . Each per mutant is defective in the ability to grow on either methanol or oleic acid, substrates that require multiple peroxisomal enzymes to be metabolized, but grows at the wild-type rate on other substrates, such as glucose, ethanol, and glycerol. Like Zellweger cells, P. pastoris per mutants lack normal peroxisomes and contain cytosolic peroxisomal matrix enzymes, e.g. catalase. In addition, alcohol oxidase, an abundant peroxisomal enzyme required for methanol metabolism, is present at greatly reduced levels in methanol-induced per mutant cells, a phenomenon that is also observed with certain peroxisomal enzymes in Zellweger cell lines. Finally, similar to Zellweger cells, methanol- or oleate-induced per cells contain abnormal single and multiple membrane-bound vesicles. We have utilized the per mutants to clone PER genes by functional complementation. We report that PER3 encodes a peroxisomal membrane protein that appears to be involved in peroxisomal protein import. Furthermore, we show that the abnormal vesicles observed in a per3 mutant contain peroxisomal matrix enzymes such as thiolase, suggesting that these structures are peroxisomal in origin.


EXPERIMENTAL PROCEDURES

Strains, Media, and Microbial Techniques

P. pastoris strains used in this study are listed in . P. pastoris cultures were grown at 30 °C in a complex medium (YPD) composed of 1% yeast extract, 2% peptone, and 2% dextrose, or in one of the following defined media: YNB medium (0.17% yeast nitrogen base, 0.5% ammonium sulfate) supplemented with either 0.4% dextrose, 0.5% methanol, or 0.2% oleate, 0.02% Tween 40, 0.05% yeast extract. Nutritional supplements for growth of auxotrophic strains were added to 50 µg/ml as required. Sporulation (mating) medium and procedures for genetic manipulation of P. pastoris have been described elsewhere (36) . P. pastoris transformations were performed as described in Cregg et al.(38) . Vector pJAH23, a gift from Dr. S. Subramani (University of California at San Diego) is composed of the firefly luciferase gene under the transcriptional control of the P. pastoris AOX1 promoter and terminator, the S. cerevisiae ARG4 gene, the P. pastoris autonomous replication sequence PARS2, and bacterial plasmid pBR322.

P. pastoris DNA Library Construction

The P. pastoris genomic DNA library used to clone PER3 was constructed in P. pastoris-Escherichia coli shuttle vector pYM8 (38) . This vector is composed of a P. pastoris autonomous replication sequence (PARS1), the S. cerevisiae histidinol dehydrogenase gene ( HIS4), which is a selectable marker for transformation of his4 strains of P. pastoris, and sequences from E. coli plasmid pBR322. To construct the library, pYM8 was digested with BamHI and treated with calf intestine alkaline phosphatase. Total DNA was extracted from wild-type P. pastoris and partially digested with Sau3AI to generate DNA fragments of 5-10 kilobase pairs (kb). Fragments in this size range were further size-selected by sucrose gradient centrifugation. Approximately equal molar amounts of Sau3AI-digested yeast DNA and BamHI-calf intestine alkaline phosphatase-treated pYM8 were ligated using T4 DNA ligase and transformed into E. coli strain MC1061 by selection for ampicillin resistance. Plasmid DNA was extracted from approximately 56,000 transformed colonies by the alkaline lysis procedure and further purified by centrifugation in an ethidium bromide/cesium chloride density gradient (39) . The library was stored as plasmid DNA in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA buffer at -20 °C.

Isolation and Characterization of PER3

P. pastoris strain JC120 ( per3-1 his4) was transformed with 5 µg of the above P. pastoris library by the spheroplast-generation CaCl-polyethelene glycol fusion method, and transformants were selected for His prototrophy on YNB dextrose medium (38) . The approximately 50,000 transformants that resulted were collected from the plates as a single pool and inoculated into liquid YNB methanol medium at an initial OD of 0.1. After 3 days, the culture grown on methanol was harvested. Total DNA was extracted from these cells and transformed into E. coli strain MC1061. Plasmid DNA was extracted from several resulting transformants and examined by cleavage with selected restriction enzymes. All recovered vectors appeared to be identical with inserts of approximately 8.1 kb. One of the vectors, pYT4, retransformed into JC120 and observed to cotransform cells to both Mut and His with approximately equal efficiency, was selected for further study. To define the location of PER3, selected subfragments from pYT4 were subcloned and tested for their ability to complement JC120 for growth on methanol. In addition, selected segments of pYT4 were removed, and the truncated vectors were religated and tested for complementation. Results indicated that the complementing activity was located within a 5-kb region between ScaI and the BamHI/ Sau3AI junction. Northern blot studies using subfragments within this region indicated that a 3.3-kb EcoRV- ScaI fragment transcribed a methanol-induced 2.1-kb mRNA. Two subfragments from this region, a 2.6-kb ScaI- HindIII fragment and a 0.7-kb XbaI- EcoRV fragment, were inserted into appropriately digested pBluescript II SK+ (Stratagene, La Jolla, CA), and a series of nested deletions was generated from each of the resulting plasmids by limited digestion with exonuclease III as described previously (39) . The DNA sequence of both DNA strands was then determined by the dideoxy method (40) using Sequenase 2.0 (U. S. Biochemical Corp., Cleveland, OH). DNA and predicted protein sequences were analyzed using MacVector software (IBI, New Haven, CT). Sequencing results revealed a long open reading frame whose 5` terminus extended beyond the EcoRV site. To complete the DNA sequence, a series of extending oligonucleotide primers was synthesized and utilized in sequencing reactions along with pYT4 DNA as template. The predicted amino acid sequence of the open reading frame was compared with those in the GenBank data base.

Construction of the PER3 Disruption Strain

A vector designed to delete most of PER3 was constructed in two steps starting with plasmid pYM25. This plasmid is composed of a 3.1-kb HindIII fragment encoding the S. cerevisiae ARG4 gene inserted into the HindIII site of pBR322. In the first step, a 2.0-kb EcoRI fragment composed of sequences flanking the 5` terminus of PER3 was inserted into the EcoRI site of pYM25 to create plasmid pYH2. Second, a 1.5-kb PvuII fragment composed of 3` PER3 sequences was inserted into the NruI site of pYH2 to create the PER3-deletion vector pYH3. The PER3 locus in the vector contains a deletion of 1638 bp of PER3 coding sequence (amino acids 1-546) with the S. cerevisiae ARG4 gene fragment inserted at the deletion site. pYH3 was linearized by partial digestion with PvuII and transformed into GS190 ( arg4). Arg transformants were selected and screened for ones that were Mut. Several independent Arg Mut transformants were collected and examined by the Southern blot method to confirm proper insertion of the linear fragment from pYH3 into the PER3 locus.

Preparation of Anti-Per3p Antibodies

Per3p was expressed in E. coli as a fusion with the maltose-binding protein (MAL) using a kit supplied by New England Biolabs. To construct the strain, the MAL expression vector, pMAL-c2, was first modified to accept a PER3-containing DNA fragment in the proper reading frame by digesting the vector with EcoRI and inserting an adaptor oligonucleotide (AATTGAATGCATTTC). The adaptor insertion resulted in the introduction of an NsiI site that was in frame with an NsiI site in PER3. A 1.0-kb NsiI- BglII fragment from pYH1 encoding amino acids 479-713 of PER3 was then inserted into NsiI- and BamHI-digested pMAL- NsiI vector to create the malE-PER3 expression vector, pEH1. MAL-Per3p fusion protein synthesis in E. coli strain TB1 transformed with pEH1 and purification were performed as recommended by the supplier.

Polyclonal antibodies against the fusion protein were raised in rabbits (Josman Laboratories, San Jose, CA). Anti-Per3p antiserum was affinity-purified by the procedure described by Raymond et al.(41) . Crude rabbit serum was passed two times through a column containing total soluble E. coli protein from induced cells of strain TB1 (pMAL-c2). The flow-through from this column was then passed two times through a column to which total soluble protein from strain JC121 ( per3::SARG4 arg4) had been bound. The flow-through from this column was collected and used at a 1:1,000 dilution.

Miscellaneous Methods

The E. coli strain used for recombinant DNA manipulations was MC1061 (39) , except for expression of malE-PER3 where strain TB1 was employed (New England Biolabs). E. coli cultivation and recombinant DNA techniques were performed as described (39) . Total protein was determined by Bradford (42) assay using bovine serum albumin as standard. Catalase (43) , acyl-CoA oxidase (44) , alcohol oxidase (45) , luciferase (46) , cytochrome c oxidase (47) , and fumarase (48) activities were measured according to published procedures. SDS-polyacrylamide gel electrophoresis and immunoblotting procedures were performed as described previously (49) . For immunoblotting experiments, rabbit polyclonal antibodies against S. cerevisiae thiolase (a gift of W.-H. Kunau, Ruhr-Universitat, Bochum, Germany), H. polymorpha catalase, and P. pastoris alcohol oxidase (a gift of M. Gleeson, SIBIA, San Diego) were used. Antigen antibody complexes were visualized using the ECL kit following the instructions of the supplier (Amersham Corp.). Electron microscopy and immunocytochemistry with rabbit antibodies raised against C. tropicalis thiolase (a gift of W.-H. Kunau, Ruhr-Universitat, Bochum, Germany) and H. polymorpha alcohol oxidase were performed as previously described (50) . Differential and discontinuous sucrose gradient centrifugations were performed as described elsewhere (5, 36) . Protein was extracted from peroxisomal membranes using either triethanolamine or carbonate as described previously (51, 52) .


RESULTS

per3 Cells Are Defective in Peroxisome Biogenesis

P. pastoris strain JC111 ( per3-1) was isolated from a mutagenized culture by screening for strains that were defective in the utilization of both methanol (Mut) and oleic acid (Out) as sole carbon and energy sources (36) . These substrates were selected because each requires several peroxisomal enzymes and functional peroxisomes to be metabolized (4) . In methanol- or oleate-grown wild-type cells, numerous large peroxisomes were readily observed (Fig. 1, A and B). In contrast, normal peroxisomes were absent from methanol- or oleate-induced per3-1 cells (Fig. 1 C). Instead, clusters of vesicular structures were frequently observed.


Figure 1: Electron micrographs showing subcellular morphology and location of selected peroxisomal enzymes in induced per3 cells. Peroxisomes in wild-type cells grown on methanol ( A) and oleate ( B). In methanol-induced per3-1 cells, peroxisomal remnants are evident ( C, arrow); these cells also contain electron-dense aggregates ( D, arrows). In methanol-induced cells of the per3 strain, peroxisomal remnants are absent ( E). Immunocytochemically, AOX protein is observed in peroxisomal remnants and cytosolic aggregates in methanol-induced per3-1 cells ( F, arrow). CAT is also found in these vesicles ( G, arrow) and in the cytosol and nucleus ( G). ( A-E, MnO-fixed cells; F-G, aldehyde/Unicryl/uranyl acetate. Abbreviations: L, lipid droplet; M, mitochondrion; N, nucleus; P, peroxisomes; V, vacuole. Bar, 0.5 µm.



Evidence for a defect in peroxisome biogenesis in per3-1 was obtained from biochemical and cytochemical experiments designed to determine the location of peroxisomal enzymes. Cell-free extracts of induced per3-1 cells were examined for activities of selected peroxisomal enzymes. In per3-1 cells induced with either methanol or oleate, catalase (CAT) activity was present at approximately the same level as wild-type cells (Fig. 2 A). In contrast, activity for alcohol oxidase (AOX), the first enzyme in methanol catabolism and a major constituent of the peroxisomal matrix of methanol-grown cells, was consistently present at levels that were less than 3% of those in wild-type P. pastoris. Initially, AOX protein levels also appeared to be low, as judged by immunoblots using anti-AOX antibodies. It was possible that AOX was synthesized in per3-1 cells but, due to inefficient import, remained mostly in the cytosol as insoluble aggregates which may have sedimented during preparation of cell-free extracts. To examine this possibility, total cell extracts were prepared without centrifugation and assayed for AOX activity and protein. Results revealed that methanol-induced per3-1 cells actually contained approximately the same amount of AOX protein as wild-type cells (Fig. 2 A). However, AOX activity levels remained low, indicating that most of the AOX protein was inactive. Further evidence of AOX aggregate formation in methanol-induced per3-1 was provided by electron microscopy studies, which showed that cytosolic aggregates were common in these cells (Fig. 1 D) and immunocytochemical experiments which showed that AOX was concentrated in these aggregates (Fig. 1 F).


Figure 2: Activity and location of selected peroxisomal enzymes in per3 strains. A, AOX and CAT activity (and AOX protein) levels in methanol-induced strains, and luciferase ( LUC) and CAT activity (and thiolase protein) levels in oleate-induced strains reported as a percentage of that in wild-type cells. Wt, wild-type P. pastoris; per3, per3-1; per3 ( PER3), per3-1 strain JC120 ( per3-1 his4) transformed with PER3-containing plasmid pYT4. B, Specific activity (or relative concentration of protein) in post-20,000 g organelle supernatant ( S) and pellet ( P) after differential centrifugation of homogenized protoplasts. ND, could not be determined due to absence of AOX activity. In immunoblots, each lane contains 10 µg of protein. Activity units are as follows: CAT, E/min/mg of protein; AOX, micromoles of product/min/mg; cytochrome c oxidase, micromoles of product/min/mg 10; LUC, arbitrary light units/mg of protein.



To determine the location of selected peroxisomal enzymes in per3-1 cells, differential centrifugation experiments were performed on homogenized spheroplasts prepared from induced cells. With either methanol- or oleate-grown wild-type cells, a large portion of CAT activity was present in the 20,000 g pellets (Fig. 2 B). In contrast, in per3-1 cells induced with either substrate, CAT was located primarily in the supernatant, indicating that the enzyme was cytosolic. As a control, cytochrome c oxidase, a mitochondrial marker enzyme, remained mostly in the pellet in per3-1 preparations. Interestingly, the small amount of AOX activity remaining in methanol-induced per3-1 cells was present in the pellet fractions (Fig. 2 B). This suggested that our per3-1 allele may not be totally defective in AOX import, and as a consequence, peroxisomes may import a small fraction of the AOX protein which then assembles into active enzyme. Evidence that a portion of AOX actually was imported was obtained by immunocytochemistry which showed that, in addition to the aggregates described above, AOX was also present within the small vesicular structures in per3-1 cells (Fig. 1 F). Identical results were also obtained for CAT (Fig. 1 G) and dihydroxyacetone synthase, the third peroxisomal enzyme in the methanol catabolic pathway (data not shown).

per3-1 Cells Are Defective in Import of Luciferase

Although it is assumed that CAT, dihydroxyacetone synthase, and AOX are all PTS1 enzymes, this has not been directly demonstrated in P. pastoris. As direct evidence that per3-1 was defective in import of PTS1 proteins, we examined the behavior of heterologously expressed luciferase, the prototypical PTS1 enzyme (10) . Wild-type and per3-1 strains were constructed that contained luciferase under control of the AOX1 promoter of P. pastoris. Both strains synthesized high levels of luciferase activity in methanol medium, moderate levels in oleate medium, and no luciferase in glucose medium (Fig. 2 A) (53) . After differential centrifugation of preparations from oleate-induced cells of these strains, luciferase activity was highly enriched in the pellet fraction from wild-type cells while activity in per3-1 cells was present mostly in the supernatant fraction (Fig. 2 B). From this, we concluded that the per3-1 strain was defective in the import of luciferase and probably other PTS1-containing proteins as well.

per3-1 Cells Import Thiolase into Peroxisome Remnants

The fate of thiolase, a peroxisomal -oxidation enzyme imported via the PTS2 pathway, was also examined in per3-1 cells (15, 16, 53) . Crude organelle pellet and supernatant fractions from oleate-induced cells were immunoblotted and reacted with antibodies against S. cerevisiae thiolase (Fig. 2 B). In contrast to CAT, AOX, dihydroxyacetone synthase, and luciferase, thiolase was concentrated in the pellet, suggesting that this PTS2 enzyme had either aggregated or was located in a subcellular compartment. To distinguish between these possibilities, the location of thiolase was examined immunocytochemically. In sections from oleate-grown wild-type cells, peroxisomes were specifically labeled by antibodies directed against C. tropicalis thiolase (Fig. 3 A). In oleate-induced per3-1 cells, the gold particles were primarily observed on the vesicular structures (Fig. 3 B), indicating that thiolase was located in these structures. Taken together, our results suggested that per3-1 was specifically impaired in the ability to import CAT, AOX, dihydroxyacetone synthase, and luciferase (PTS1 enzymes) but not thiolase (a PTS2 enzyme), which appeared to be efficiently imported. Furthermore, the presence of thiolase and other peroxisomal enzymes in the vesicular structures indicates that these structures represent peroxisomal remnants.


Figure 3: Immunocytochemical labeling of oleate-induced cells with anti-thiolase antibodies. Thiolase labeling is concentrated in peroxisomes in wild-type cells ( A) and in vesicles in per3-1 cells ( B, arrow), but is located in cytosol and nucleus in per3 cells ( C). Methanol-grown cells of the per3-1 strain complemented with PER3-containing plasmid pYT4 contain normal peroxisomes with AOX protein in the organelles ( D). Bar, 0.5 µm.



Isolation and Characterization of PER3

The PER3 gene was isolated from a P. pastoris genomic DNA library by functional complementation of per3-1 strain JC120 ( per3-1 his4). Transformants were initially selected for histidine prototrophy (His) on glucose medium and subsequently selected for growth on methanol (Mut). Total DNA was extracted from a pool of His Mut transformants, and plasmids were recovered by transforming the DNA into E. coli. A plasmid, designated pYT4 (Fig. 4 A), was isolated that co-transformed the per3-1 his4 strain to both His and Mut simultaneously. per3-1 cells transformed with pYT4 contained normal-appearing peroxisomes (Fig. 3 D). In addition, AOX activity in methanol-grown transformants was restored to normal levels and both AOX and CAT were again present in peroxisomes (Figs. 2 and 3 D). Restriction mapping of pYT4 revealed a P. pastoris DNA insert of approximately 8.1 kb. By subcloning selected subfragments from pYT4, the complementing activity was located within a region of 5.0 kb. Northern blots using selected fragments from this region revealed a transcript of approximately 2.1 kb that was present at low levels in glucose-grown wild-type P. pastoris. Relative to total RNA levels, this transcript appeared to be induced approximately 5-fold in methanol- or oleate-grown cells (Fig. 5). Since peroxisomes are also induced by these substrates, the higher level of this transcript was consistent with it being the product of a gene coding for a peroxisomal protein. The DNA sequence of the region revealed an open reading frame (ORF) of 2,139 bp with the potential of encoding a polypeptide of 713 amino acids (81 kDa) (Fig. 6 A). Further studies indicated that this ORF was PER3. As described below, a P. pastoris strain in which most of the ORF was deleted was peroxisome-deficient and was determined genetically to be an allele of per3-1. In addition, the product of the ORF was found to be a peroxisomal protein.


Figure 4: Diagrams of plasmid pYT4 and the PER3-deletion allele per3. A, restriction map of the PER3 locus cloned in pYT4. The asterisk marks a HindIII site in the PER3 gene that was used for subcloning but is not unique to the vector. B, the structure of the per3 allele. To construct this allele, PER3 DNA sequences encoding Per3p amino acids 1 through 546 were replaced with a DNA fragment containing the S. cerevisiae ARG4 gene and inserted into the P. pastoris genome by homologous recombination. C, correct targeting of the per3 fragment was demonstrated by Southern blotting of genomic DNAs cut with ClaI and hybridizing with a labeled fragment ( P) indicated in B. Lane 1, 2 µg of wild-type genomic DNA; lane 2, 2 µg of DNA from the per3 strain JC121.




Figure 5: Northern blot of PER3 message. All lanes contain 10 µg of total RNA. Odd-numbered lanes contain RNAs from glucose ( G)-grown cells. Even-numbered lanes contain RNAs from methanol ( M)-induced cells. Lanes 1 and 2, wild-type P. pastoris; lanes 3 and 4, per3; lanes 5 and 6, per3-1. As a control, the filter was also hybridized with a labeled DNA fragment encoding the S. cerevisiae actin gene ( ACT). ACT message levels are significantly lower in methanol-grown wild-type P. pastoris cells than in glucose-grown cells. Levels are even lower in methanol-induced cells of the per3 mutants.




Figure 6: PER3 sequence. A, nucleotide and predicted amino acid sequences of PER3. Potential -helical membrane-spanning domains are underlined. B, hydrophilicity plot of Per3p-predicted primary structure shows that the peptide is hydrophobic in overall character. These sequence data are available from EMBL/GenBank/DDJB under accession no. L40485.



Hydropathy analysis indicated that the predicted PER3 product (Per3p) was hydrophobic in overall character with at least three potential -helical transmembrane domains (Fig. 6, residues 338-358, 373-392, and 637-660) (54) . Two additional regions with lower but significant transmembrane potential were also identified (Fig. 6, residues 484-510 and 522-540). The primary sequence of Per3p showed significant similarity to that of Per1p, a protein required for peroxisome biogenesis in H. polymorpha(20) (see ``Discussion''). Data base searches revealed no other proteins with overall sequence similarity to Per3p. The only identifiable sequence feature of Per3p was the three carboxyl-terminal amino acids (Ala-Lys-Leu-COOH), a known PTS1 sequence variant.

Per3p Is a Peroxisomal Membrane Protein

Per3p was characterized through rabbit polyclonal antibodies raised against Per3p expressed in E. coli as a fusion with the maltose-binding protein. In immunoblots prepared from methanol- or oleate-grown wild-type extracts, affinity-purified anti-Per3p antibody preparations recognized an approximately 76-kDa polypeptide that was not present in extracts from methanol- or oleate-induced cells of a strain in which most of PER3 had been deleted ( per3) (Fig. 2 B). The apparent molecular mass of Per3p was somewhat less than the 81-kDa mass calculated from the predicted primary sequence. This discrepancy could be due to the high content of nonpolar amino acids in Per3p (42%) which can cause proteins to migrate faster in SDS-polyacrylamide gel electrophoresis (55) . Per3p was present at a significantly higher level in methanol- and oleate-grown cells than in glucose-grown cells (not shown), a result that was consistent with our observations of PER3 mRNA and the induction of peroxisomes under these growth conditions.

As a first step in establishing the subcellular location of Per3p, methanol- and oleate-grown wild-type P. pastoris cells were subjected to differential centrifugation as described above, and the resulting pellet and supernatant fractions were examined for Per3p by immunoblotting. Per3p was present primarily in the organelle pellet along with a large portion of CAT (Fig. 2 B). A crude organelle pellet from oleate-grown cells was then layered over a discontinuous sucrose gradient and centrifuged to separate peroxisomes from other organelles (mainly mitochondria). In these gradients, peroxisomes were concentrated at a density of 1.21 g/cm as judged by the location of CAT and acyl-CoA oxidase activities, and thiolase protein (Fig. 7 A, fraction 6). Mitochondria were located at a density of 1.16 g/cm as judged by fumarase activity (Fig. 7 A, fraction 12). Per3p was found exclusively in the peroxisomal fraction, indicating that the protein is peroxisomal. Samples of purified peroxisomes from these fractions were extracted with either triethanolamine or sodium carbonate. Following triethanolamine treatment and centrifugation at 30,000 g, CAT and thiolase were located mostly in the supernatant, whereas Per3p was in the pellet, suggesting that Per3p was associated with the peroxisomal membrane (Fig. 7 B, lanes 2 and 3). After carbonate extraction and centrifugation at 200,000 g, most Per3p remained in the membrane pellet, suggesting that Per3p may be an integral peroxisomal membrane protein (Fig. 7 B, lanes 4 and 5). However, we repeated the carbonate treatment using crude organelle pellet fractions from methanol- and oleate-induced cells and found that, in contrast to the results with purified peroxisomes, only about 25% of Per3p was resistant to extraction (data not shown). Similar results have been obtained with the S. cerevisiae 24-kDa peroxisomal membrane protein (Pmp24p) where the inextractibility of the protein from purified peroxisomes was believed to be an artifact of the extensive manipulations involved in the isolation of peroxisomes.() We conclude that Per3p is tightly associated with the peroxisomal membrane but is probably not an integral membrane protein.


Figure 7: Per3p is a peroxisomal membrane protein. A, sucrose density gradient profile of crude organelle pellet fraction derived from oleate-induced wild-type P. pastoris cells. Activity in each fraction is presented as a percentage of total activity in gradient. Fraction 1 ( left) is gradient bottom and fraction 25 ( right) is gradient top. ACO, acyl-CoA oxidase activity. Immunoblotting was performed with equal volumes (200 µl) of selected gradient fractions. Total protein was visualized by staining with Coomassie Blue dye. B, in lanes 1 through 5, 25 µg of purified peroxisomes isolated from oleate-grown wild-type cells ( lane 1) were extracted with 20 mM triethanolamine, pH 7.8 ( lanes 2 and 3) or 0.1 M sodium carbonate, pH 11 ( lanes 4 and 5). After extraction, samples were centrifuged, and supernatant ( even-numbered lanes) and pellet ( odd-numbered lanes) fractions were immunoblotted with anti-Per3p antibodies.



A PER3 Deletion Strain Lacks Peroxisomes and Contains Cytosolic Thiolase

A P. pastoris strain in which most of PER3 was deleted was created by the gene replacement method (56) . For the replacement, a plasmid was constructed in which 1638 bp of PER3 coding sequence (nucleotides 1 through 1638 encoding amino acids 1 through 546 in Fig. 6 A) were removed and replaced with a fragment containing the S. cerevisiae ARG4 gene. This plasmid was then digested with a restriction enzyme to release the PER3 deletion allele ( per3) on the linear DNA fragment, shown in Fig. 4B, and introduced into P. pastoris strain GS190 ( arg4). Transformants in which the fragment had deleted the PER3 locus were isolated by selecting for Arg colonies and then screening for ones which were also Mut. Proper targeting of the fragment was confirmed by Southern blot analysis (Fig. 4 C). In addition, Northern blots showed that methanol-induced cells of the per3 strains no longer produced the 2.1-kb PER3 message (Fig. 5 B, lanes 3 and 4). One per3-derived strain, JC121 ( per3::SARG4 arg4), was crossed with JC120 ( per3-1 his4) by selection for growth on minimal glucose plates. The resulting diploids were tested for methanol growth and were Mut. Several thousand spore products from these diploids were then examined by replica plating for Mut phenotype and all were Mut as well, demonstrating that the per3-1 and per3 alleles were tightly linked and likely to be mutant alleles of the same gene.

Phenotypically, the per3 strain was clearly a per mutant. In addition to being Mut and Out, electron microscopy examination of methanol-induced per3 cells showed that they lacked intact peroxisomes, and biochemical studies showed that CAT was induced to normal levels in both methanol- and oleate-induced cells but was located in the cytosol (Fig. 2). However, the per3-1 and per3 strains were different in several respects. First, methanol-induced cells of per3-1 frequently contained clusters of peroxisomal remnants, which were absent in methanol- or oleate-induced per3 cells (Fig. 1 E). Thus, the phenotype of a per3 null mutant strain appears to be the complete absence of peroxisomes. Second, methanol-induced per3 cells contained no AOX activity, whereas per3-1 cells contained low but significant amounts of sedimentable AOX activity (Fig. 2, A and B). These results support our suggestion that the per3-1 allele in the mutant strain is slightly leaky with respect to AOX import and that active AOX enzyme is solely present in peroxisomal remnants. Third, thiolase was found biochemically (Fig. 2 B) and immunocytochemically (Fig. 3 C) to be located in the cytosol. These results were in contrast to induced per3-1 cells where thiolase was located primarily within the peroxisomal remnants (Figs. 2 B and 3 B). Thus, although both per3-1 and per3 were deficient in ability to import CAT, AOX, and luciferase, per3-1 cells selectively imported thiolase while per3 cells did not.


DISCUSSION

Previously we described the isolation and partial characterization of a collection of P. pastoris mutants defective in peroxisome biogenesis ( per mutants) (36, 37) . In this study, we report the detailed analysis of mutants in PER3 as well as the isolation and characterization of PER3 and its product Per3p. The primary sequence of Per3p predicts a 713-amino acid protein that is hydrophobic in overall character with three potential -helical transmembrane domains. Consistent with these predictions, biochemical studies showed that Per3p has an apparent molecular mass of approximately 76 kDa, is tightly associated with the peroxisomal membrane, and may even be an integral membrane protein of the organelle. Per3p terminates in the tripeptide sequence Ala-Lys-Leu-COOH (AKL), a variant within the PTS1 group that is sufficient to target reporter proteins to peroxisomes (11, 20) . The presence of a PTS1 on Per3p is surprising since this targeting signal is thought to be responsible for import of matrix proteins and not membrane proteins (14) . Other peroxisomal proteins that specifically end in AKL include: mammalian sterol carrier 2 (57) , glycosomal glyceraldehyde phosphate dehydrogenase of Trypanosoma brucei(58) , acetoacetyl-CoA thiolase A of C. tropicalis(59) , PMP20 of C. boidinii(60) , and Per1p of H. polymorpha(20) . However, only a few peroxisomal membrane proteins have been described and it is not yet clear whether PTS1 participates in the targeting of some membrane proteins. It will be interesting to determine whether the motif on Per3p is a functional targeting signal and whether it is responsible for targeting Per3p to the peroxisomal membrane.

Data base searches revealed only one other protein with significant sequence similarity to Per3p. Per1p, a protein required for peroxisome biogenesis in the methylotrophic yeast H. polymorpha, is 42% identical and 60% similar to Per3p, and may be its functional homologue (Fig. 8) (20) . Despite their similarity, differences between the proteins are evident. First, Per1p is significantly smaller than Per3p (71 versus 81 kDa) and contains a functional PTS2 sequence not present in Per3p. Second, P. pastoris and H. polymorpha are closely related species (61) , and other proteins show a much greater degree of similarity between these two yeast species. For example, the primary sequences for AOX and dihydroxyacetone synthase are each greater than 80% identical (62, 63, 64) .() Thus, it might be expected that the H. polymorpha homologue of Per3p would show greater similarity than Per1p. Second, Per3p is a membrane protein while Per1p is located in the peroxisomal matrix (20) . Third, the H. polymorpha PER1 gene does not complement per3 mutants of P. pastoris, and PER3 does not complement H. polymorpha per1.() In contrast, the H. polymorpha genes for AOX and formaldehyde dehydrogenase (another methanol pathway-specific enzyme) readily complemented P. pastoris mutants in these genes, and Northern blots indicated that transcription of the H. polymorpha genes initiates correctly and is properly regulated. Thus, it is possible that Per3p and Per1p are not homologues but are different members of a family of related proteins required for peroxisome biogenesis.


Figure 8: Comparison of the predicted amino acid sequences of P. pastoris Per3p and H. polymorpha Per1p. Sequences were aligned using PC gene software. The character ``'' between sequences indicates residues that are identical. The character ``.'' indicates similar residues. Similar residues are defined as: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.



Our studies of per3-1, a nitrosoguanidine-generated mutant, show that the numerous vesicular structures frequently observed in induced cells of this strain are peroxisomal in origin. In oleate-induced per3-1 cells, these vesicular structures contain thiolase but only minor amounts of PTS1 proteins, while the bulk of these enzymes is present in the cytosol. This is the first direct visual evidence that these structures, which are observed in most P. pastoris per mutants, are remnants of peroxisomes in P. pastoris. In Zellweger cell lines, similar peroxisomal remnants, called peroxisomal ghosts, are also observed (32) . The additional layer(s) of membrane that often surround peroxisomal remnants may be the result of their inclusion within autophagic vacuoles (65) . In methylotrophic yeasts, cells shifted from methanol to other carbon sources are known to rapidly sequester peroxisomes within autophagic structures where they are degraded by vacuolar enzymes (66) . Furthermore, in Zellweger cells, a significant portion of the peroxisomal ghosts are enclosed within compartments that contain lysosomal proteins, suggesting that they are subject to rapid turnover by autophagy as well (67) .

Our data suggest that Per3p plays an essential role in matrix protein import. This conclusion is based on our observation that the peroxisomal remnant structures in induced per3-1 cells efficiently import thiolase but not AOX, CAT, dihydroxyacetone synthase, or luciferase. Thiolase is imported via a PTS2 pathway in mammals and yeast (15, 16) , whereas luciferase is imported via a PTS1 pathway (10) . Although PTSs for AOX, CAT, and dihydroxyacetone synthase have not been defined in P. pastoris, their counterparts in H. polymorpha have been shown to be PTS1 enzymes (68, 69) and, therefore, are likely to be imported via a PTS1 pathway in P. pastoris as well. Thus, per3-1 cells are primarily affected in PTS1 but not PTS2 import. The per3-1 strain is not totally defective in PTS1 import, since small amounts of CAT and AOX are found in the peroxisomal remnant structures. Interestingly, upon differential centrifugation of methanol-induced per3-1 preparations, virtually all of the active AOX enzyme is found in the crude organelle pellets, indicating that only AOX which gains entry into a peroxisome assembles into active octameric enzyme, while the majority of AOX remains in the cytosol as inactive aggregates. That only imported AOX becomes active suggests that AOX assembly may require the presence of one or more peroxisomal factors such as the AOX co-factor FAD or a peroxisomal assembly factor (70) .

An additional clue to Per3p function came from studies comparing the per3-1 strain to a strain deleted for most of PER3 ( per3). Although neither per3 mutant can grow on methanol or oleate and both are peroxisome deficient, they display at least two differences. First, in induced per3 cells, we cannot find the peroxisomal remnant structures that are so prevalent in per3-1. Second, thiolase is in the cytosol in oleate-induced per3 cells, whereas it is in the peroxisomal remnants in per3-1 cells. Thus, the per3 null phenotype appears to be an inability to import either PTS1- or PTS2-containing proteins.

To explain the per3 mutant phenotypes, we propose that Per3p is an essential component of the cell's machinery for import of both PTS1 and PTS2 (and perhaps all) matrix proteins. To account for the continued ability of per3-1 cells to import thiolase, we suggest that Per3p is composed of several functional domains and that in the per3-1 allele, a mutant product is synthesized that is defective in a domain primarily involved in the import of PTS1 proteins but not PTS2 proteins. Consistent with partially functional Per3p, we observe that the mutant protein is present in induced per3-1 cells and that it is the same size as in wild-type cells (Fig. 2 B). To explain the phenotype of the per3 strain, we suggest that other domains in Per3p are essential for PTS2 protein import (or both PTS1 and PTS2 import); therefore, deletion of PER3 results in a strain that is defective in all matrix protein import.

The identification of two PTSs for peroxisomal protein import has raised the question of whether cells also have two independent peroxisomal translocation pathways or only have separate PTS receptors but otherwise share most components of a common translocation machinery (53, 71) . Despite the large numbers of peroxisome-deficient yeast mutants that have been characterized, only two appear to be selectively defective in a specific import pathway. P. pastoris pas8 ( S. cerevisiae pas10; H. polymorpha per3) mutants are specifically defective in PTS1 protein import and the Pas8p has been shown to specifically bind a peptide ending in the PTS1 sequence, Ser-Lys-Leu-COOH (53, 72) .() Thus, Pas8p appears to be the PTS1 protein receptor. Conversely, S. cerevisiae pas7 mutants appear to be selectively defective in PTS2 import (73) . (Similar S. cerevisiae mutants have been reported as peb5 and peb1 and may be alleles of pas10 and pas7, respectively (71) .) The small number of pathway-specific mutants is most easily explained as a consequence of a shared import pathway. Our model of Per3p function in which the protein is composed of domains preferentially involved in import of different PTS protein classes also appears to be most consistent with a shared import pathway model.

  
Table: Yeast strains



FOOTNOTES

*
This research was supported by National Science Foundation Grant MCB-9118062 and National Institutes of Health Grant DK-43698 (to J. M. C.). 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.

§
To whom correspondence should be addressed: Dept. of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, P.O. Box 91000, Portland, OR 97291-1000. Tel.: 503-690-1217; Fax: 503-690-1464; E-mail: cregg@admin.ogi.edu.

The abbreviations used are: PTS, peroxisomal targeting signal; AOX, alcohol oxidase; CAT, catalase; MAL, maltose-binding protein; Mut, methanol-utilizing; Mut, methanol-utilization-defective; ORF, open reading frame; Out, oleate-utilization defective; bp, base pair; kb, kilobase pair.

J. Goodman, personal communication.

G. Thill, personal communication.

J. Cregg, unpublished results.

M. Veenhuis, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to Ineke Keizer-Gunnink and Klaas Sjollema for technical assistance with the electron microscopy experiments. We thank Dr. W.-K. Kunau for antibodies against S. cerevisiae and C. tropicalis thiolases, and Dr. S. Subramani for the AOX1p-LUC plasmid pJAH23. We acknowledge Dr. H. Waterham for helpful discussions and critical reading of this manuscript.


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