©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Hansenula polymorpha PER3 Gene Is Essential for the Import of PTS1 Proteins into the Peroxisomal Matrix (*)

(Received for publication, February 27, 1995)

Ida J. van der Klei (§) , Reinder E. Hilbrands , Gert Jan Swaving (¶) , Hans R. Waterham (**) , Engel G. Vrieling , Vladimir I. Titorenko , James M. Cregg (1)(§§), Wim Harder , Marten Veenhuis

From the Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands and theOregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

PER genes are essential for the assembly of peroxisomes in Hansenula polymorpha. Here we describe the PER3 gene which was cloned by functional complementation of a H. polymorpha per3 mutant. The complementing PER3 gene encodes a protein of 569 amino acids (Per3p) with a calculated mass of 63.9 kDa; Per3p belongs to the tetratricopeptide repeat protein family and is located in both the cytosol and the peroxisomal matrix. Remarkably, Per3p does not contain a known targeting signal (PTS1 or PTS2). The PER3 gene product shows similarity to the Saccharomyces cerevisiae Pas10p (40% identity) and the Pichia pastoris Pas8p (55% identity). However, their function apparently cannot be interchanged since the P. pastoris PAS8 gene failed to functionally complement a H. polymorpha per3 disruption mutant.

The per3 disruption mutant contained normal but small peroxisomes in which PTS2 proteins (both homologous and heterologous) were imported. Other matrix proteins (in particular PTS1 proteins) resided in the cytosol where they were normally assembled and active.

We argue that Per3p is a component of the peroxisomal import machinery and most probably shuttles matrix proteins from the cytosol to the organellar matrix.


INTRODUCTION

Peroxisomes are important cell organelles, which carry out various metabolic functions in eukaryotic cells (for a review, see (1) ). The organelles do not contain DNA and lack an independent protein synthesizing machinery; hence all matrix proteins are synthesized in the cytosol and subsequently sorted to the organelle (for a review, see (2) ). Two types of peroxisomal targeting signals (PTS)()have been identified, namely PTS1 (located at the extreme C terminus) and PTS2, present at the N terminus and characterized by the consensus sequence RL-X-H/QL(3, 4) . Both PTS1 and PTS2 are conserved among higher and lower eukaryotes (5, 6) , and either one is necessary and sufficient for targeting of homologous or heterologous proteins to the peroxisomal matrix.

Components essential for peroxisome biogenesis have been identified from an analysis of various yeast peroxisome assembly mutants (for a review, see (2) ). The biochemical phenotype of some of these mutants led to the assumption that in yeasts separate import pathways exist for PTS1 and PTS2 proteins(7, 8, 9) .

In our laboratory, we use the methylotrophic yeast Hansenula polymorpha as a model organism for studies on peroxisome biogenesis and function(10) . In this organism several PTS2 proteins have been identified (e.g. amine oxidase(11) , thiolase and Per1p(12) ). Recently, we have demonstrated that the PTS2 import mechanism is conserved because heterologous PTS2 proteins like watermelon gMDH (6) and Saccharomyces cerevisiae thiolase (13) are normally sorted to H. polymorpha peroxisomes.

In the present study, we provide further evidence that a separate PTS2 import machinery exists in H. polymorpha. This was evident from studies on one of the H. polymorpha import (Pim) mutants (14) belonging to the per3 complementation group. In a per3 deletion mutant, small peroxisomes were present which were capable to import PTS2 proteins, while other matrix proteins (e.g. PTS1 proteins) remained in the cytosol. From these data we conclude that Per3p is a component of a peroxisomal protein import machinery which is redundant for PTS2 proteins.


EXPERIMENTAL PROCEDURES

Organisms and Growth Conditions

The H. polymorpha strains used are wild type (WT) CBS4732, the NCYC495 derived leu1.1, leu1.1 ura3 and per3-188 leu1.1, per3-191 leu1.1, per3-229 leu1.1 and per3-237 leu1.1(14, 15) . H. polymorpha was grown at 37 °C on rich complex medium (YPD) containing 1% yeast extract, 2% peptone, and 1% glucose, on minimal media as described (16) or on YNB containing 0.67% yeast nitrogen base (Difco). The carbon sources used are 0.5% glucose, 0.5% ethanol, 0.5% methanol, or 0.1% (v/v) oleic acid + 0.02% (v/v) Tween-80; the nitrogen sources used are ammonium sulfate or (m)ethylamine at 0.2%. Carbon-limited continuous culturing was carried out as described previously (16) at a dilution rate of 0.1 h using 0.5% glucose and 0.2% choline. Amino acids and uracil were added to a final concentration of 30 µg/ml.

Escherichia coli strains MC1061, C600, and DH5 were grown at 37 °C in LB medium or in minimal M9 medium (17) supplemented with ampicillin (50 µg ml) or kanamycin (60 µg ml), when appropriate.

Cloning and Characterization of the PER3 Gene

per3-237 was transformed with a genomic DNA library of H. polymorpha(12) . Leucine prototrophs were replica-plated onto YNB plates containing 0.5% methanol and screened for the ability to grow on methanol. E. coli MC1061 was transformed by DNA isolated from complemented cells, and a plasmid containing a genomic insert of approximately 20 kb was recovered. Retransformation of per3-237 with this plasmid resulted again in restoration of growth on methanol. The 20-kb insert was partially digested with Sau3A, and resulting fragments were cloned in the unique phosphatase-treated BamHI site of the pHRP2 vector(18) . pHRP2 plasmids containing the complementing insert were selected by another round of complementation and rescue in E. coli. All complementing plasmids contained a genomic insert of 4.7 kb, which was subcloned in two orientations in pOK12 (19) . Nested sets of deletions were generated using exonuclease III (17) . Subclones were sequenced using the reverse primer (Stratagene). Double-stranded sequencing was performed in two directions by the dideoxy method(20) . For analysis of the DNA and amino acid sequence the PC-GENE program release 6.70 (IntelliGenetics Inc.) was used. The amino acid sequence was compared with the GenBank(R) Release 84.0.

Plasmid Constructions and Miscellaneous Genetic Methods

A BglII-HindII 3.4-kb fragment containing the PER3 gene and approximately 1.4 kb promoter region was cloned into the BamHI and NheI (blunt) sites of pHARS1(21) . The same sites of pHARS1 were used for the cloning of the NotI (blunt)-BglII fragment containing the alcohol oxidase promoter (P) and the Pichia pastoris PAS8 gene. The latter fragment was constructed by cloning the BglII (blunt)-XmaI PAS8 fragment from pSP72-PAS8 (a gift from Dr. S. Subramani) into the HindIII (blunt) and XmaI sites of pHIPX4(22) .

For overexpression PER3 was cloned into pHIPX4 behind the P. HindIII and NdeI sites were introduced upstream the PER3 start codon using polymerase chain reaction. A 2.1-kb NdeI (blunt)-SacI fragment was cloned into the HindIII (blunt)-SacI sites of pHIPX4.

The BamHI-NheI fragment of pGF159(6) , containing the gene encoding the precursor of watermelon (Citrullus vulgaris) gMDH downstream of the P was also cloned into plasmid pHARS1.

Standard recombinant DNA techniques, E. coli transformation, and plasmid isolation were performed as described(17) . H. polymorpha was transformed by electroporation(22) . Mating and random spore analysis were carried out as described(15) .

PER3 Disruption

A gene disruption construct was made by blunt-ended cloning of the 2.2-kb EcoRI-BamHI fragment of pMK155, containing the LEU2 gene of Candida albicans (obtained from Dr. E. Berardi, Ancona, Italy) in the ApaI-MscI sites of the complementing fragment. The LEU2-containing insert was subsequently released by digestion with HindIII and MluI and linearly transformed to H. polymorpha NCYC 495 ura3 leu1.1. Leucine prototrophic transformants which were methanol-utilization-deficient were checked for the proper insertion into the genome by Southern analysis (data not shown). The per3::LEU2 could be complemented with pHARS containing the 3.4-kb PER3 fragment.

Biochemical Methods

Crude extracts were prepared as described(14) . Cell fractionation was performed as described(23) , except that 1 mM phenylmethylsulfonyl fluoride and 2.5 µg/ml of leupeptine were added to all solutions. Peroxisomal peak fractions were subjected to carbonate extraction(24) . Protein concentrations were determined as described (25) using bovine serum albumin as standard. SDS-polyacrylamide gel electrophoresis was performed as described(26) . Gels were used for Western blotting(27) , and the blots were decorated using the Protoblot Immunoblotting system (Promega Biotec) and specific polyclonal antibodies against H. polymorpha peroxysomal proteins.

Generation of Per3p Antibodies

The Protein Fusion System (New England Biolabs) was used for overexpression of a maltose-binding protein-Per3 fusion protein in E. coli. A BstXI (blunt)-EcoRI fragment, encoding Per3p except for the first 15 amino acids, was cloned in frame behind the malE gene in the pMAL-P2 vector. The maltose-binding protein-PER3 protein was recovered from inclusion bodies, subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto nitrocellulose. The maltose-binding protein-Per3p band was cut out, dissolved in MeSO, and upon dilution with phosphate-buffered saline used to immunize a rabbit. The antiserum was affinity purified by incubation with crude extracts from per3 coupled to Sepharose-6B prior to use(28) .

Electron Microscopy

Whole cells and spheroplasts were fixed and embedded in Epon 812 or Unicryl(12) . Cytochemical staining of alcohol oxidase and amine oxidase activities were performed by the methods described previously(29) . Ultrathin Unicryl sections were labeled using polyclonal antibodies raised in rabbit and goat-anti-rabbit antibodies conjugated to gold according to the instructions of the manufacturer (Amersham). Freeze etching was performed as described previously(12) .

Immunofluorescence

Intact cells were prefixed for 2 h at room temperature in 3% (v/v) formaldehyde in 40 mM potassium phosphate buffer, pH 6.5, and subsequently converted into protoplasts(30) . Immunofluoresence was performed using anti-amine oxidase, affinity purified anti-Per3p antibodies and anti-rabbit fluorescein isothiocyanate(30) .


RESULTS

PER3 Encodes a Protein Belonging to the TPR Protein Family

The PER3 gene was cloned by functional complementation of a per3 mutant (per3-237), using a H. polymorpha genomic library and restoration of growth on methanol as a selection criterion. Sequence analysis of the complementing fragment revealed a 1.7-kb open reading frame, encoding a protein of 569 amino acids with a calculated mass of 63.9 kDa (Fig. 1). In the PER3 gene product (Per3p) no membrane-associated helices are predicted. The protein shows similarity to the P. pastoris Pas8p (55% identity; 8) and the S. cerevisiae Pas10p (40% identity; 9), which both are essential for import of many matrix proteins (in particular PTS1 proteins) into peroxisomes. The highest similarity exists in the C-terminal part of the protein, which contains a tetratrico peptide repeat (TPR) motif, characterized by a highly degenerate 34 amino acid repeat(31) . In Per3p seven of these repeats were found.


Figure 1: Nucleotide and amino acid sequence of the H. polymorpha PER3 gene. The amino acids are shown in one-letter code below the second nucleotide of each codon. The nucleotides are numbered on the left, the amino acid residues on the right. The region of the TPR motif is underlined.



A mutant in which most of the PER3 gene is deleted was constructed by replacing the region surrounding the translation initiation site. The resulting mutant (per3) was unable to grow on methanol and contained large cytosolic alcohol oxidase crystalloids, which are characteristic for constitutive H. polymorpha per mutants(32) . Growth on methanol and normal peroxisome development was restored upon transformation of per3 by a 3.4-kb fragment, containing the open reading frame of PER3 or by a plasmid containing PER3 under the control of the P (see below).

Upon mating of per3 (ura3 leu1 per3::LEU2) with an auxotrophic WT strain (leu1 PER3) and subsequent random spore analysis, monogenic segregation was found for both Leu and Mut phenotypes, whereas the LEU2 gene invariably co-segregated with the Mut phenotype. Diploids obtained after crossing per3 with original per3 mutants carrying four different alleles (per3-188, per3-191, per3-229, and per3-237) all displayed the Mut phenotype. Linkage analysis of two of the hybrids revealed no meiotic segregants with a recombinant Mut phenotype (384 segregants of each hybrid were tested). Taken together, these data indicate that the authentic PER3 gene was cloned.

The per3 could not be functionally complemented by the P. pastoris PAS8 gene, although few small peroxisomes were formed in the transformants. Immunocytochemical studies revealed that the Pas8p was present in the cytosol (data not shown), which is different from the location of Per3p (see below). Whether the S. cerevisiae PAS10 gene is able to complement per3 has no been tested so far.

Peroxisome Biogenesis Is Not Impaired in the PER3 Disruption Mutant

In order to gain more insight in the function of the Per3p, we analyzed the phenotype of per3 in detail. As observed for all other H. polymorpha per mutants, per3 displayed a Mut phenotype; however, the cells grew well on complex media like YPD and on mineral media containing various carbon (glucose, glycerol, and ethanol) or nitrogen sources (ammonium sulfate and primary amines). Under these conditions generally one or a few small peroxisomes were observed per cell (Fig. 2A). This indicates that Per3p is not essential for peroxisome biogenesis.


Figure 2: Panel A shows a detail of a cell of per3 grown in batch culture on ethanol-ethylamine which characteristically contains one peroxisome. Numerous small peroxisomes are present when per3 cells are grown in a carbon-limited chemostat on glucose-choline (panel B). In addition membrane protrusions are evident; a magnification of these is shown in panel C. These membranes (panel D, arrow) are also evident in freeze-fractured cells and thus do not represent artifacts due to the fixation procedure. In per3 cells incubated in oleic acid-containing media, numerous membranous layers also typically develop, associated with one or few peroxisomes (panel E). Panel F shows the presence of many normal peroxisomes in cells of the complemented per3 strain. The abbreviations used are: N, nucleus; P, peroxisome; V, vacuole. Cytosolic alcohol oxidase crystalloids are indicated by an asterisk (*). The marker represents 0.5 µm.



The per3 cells were subsequently grown in glucose-limited chemostat cultures in the presence of choline as the sole nitrogen source. In WT cells these growth conditions cause massive proliferation of peroxisomes and a high level induction of various peroxisomal matrix enzymes(32) . In H. polymorpha choline is first converted to trimethylamine which is subsequently metabolized via dimethylamine into methylamine and two formaldehyde molecules(33) . Methylamine in fact serves as the actual nitrogen source under these growth conditions and is metabolized by peroxisomal amine oxidase into formaldehyde, hydrogen peroxide, and ammonium ions. Because glucose is provided at a growth-limiting rate in these chemostat cultures, peroxisomal enzymes involved in methanol metabolism are highly expressed due to these derepressing conditions together with the strong induction by formaldehyde, released from choline utilization. These peroxisomal enzymes include alcohol oxidase, catalase, and dihydroxyacetone synthase(32) , and each reach levels equal to those obtained in methanol-limited WT cultures.

In per3 cells grown in glucose-choline chemostat cultures, many small peroxisomes developed. These organelles were frequently observed in conjunction with membranous protrusions. These membranous structures were also observed in freeze etch replica's of spray-frozen cells, which implies that they are not an artifact of the chemical fixation procedure. The architecture of these membranes resembles those of peroxisomes in WT cells in that they show smooth fracture faces which are largely devoid of proteinaceous particles (Fig. 2, B-D). This morphological phenotype suggests that the biosynthesis of the peroxisomal membrane is not disturbed or reduced in per3 cells. This was also indicated from the morphology of per3 cells incubated in oleic acid-containing medium. Unlike S. cerevisiae and P. pastoris, H. polymorpha cannot grow on this compound. However, upon incubation of WT cells in oleic acid-containing medium, many peroxisomal enzymes are synthesized, including enzymes of the -oxidation pathway and methanol metabolism; these enzymes are present in a peroxisomal compartment which is associated with excessive amounts of membranous layers(34) . Upon incubation of per3 in oleic acid-containing medium, similar structures developed, indicating again that peroxisome biosynthesis was not disturbed in the mutant cells (Fig. 2E). However, in contrast to WT cells, these organelles lacked the typical alcohol oxidase crystalloids which were instead located in the cytosol (data not shown). Therefore, these data suggest that Per3p may be involved in matrix protein translocation across these membranes.

Most Peroxisomal Matrix Proteins Are Located in the Cytosol of per3

We tested the location of various peroxisomal enzymes in cells of per3 grown in chemostat cultures on glucose-choline. After fractionation of homogenates prepared from these cells, the PTS1 proteins alcohol oxidase and catalase were almost exclusively found in the soluble fraction, suggesting a cytosolic location. Also malate synthase, which contains neither a PTS1 nor a PTS2(35) , was mainly found in the soluble fraction. In contrast, amine oxidase, a PTS2 protein(11) , was clearly pelletable (Fig. 3A).


Figure 3: Subcellular location of peroxisomal proteins in glucose-choline chemostat cells (A) or ethanol-ethylamine grown batch cells (B) of per3. Cell homogenates were fractionated by differential centrifugation resulting in an organellar pellet and supernatant fraction. The organellar pellet was subjected to sucrose density centrifugation. Equal amounts of protein from the organellar pellet (1), the high speed supernatant (2), and the peroxisomal peak fractions (3) of the sucrose gradient were loaded per lane and subjected to SDS-polyacrylamide gel electrophoresis and Western blotting using antibodies raised against different peroxisomal matrix proteins as indicated. AMO, amine oxidase; AO, alcohol oxidase; Cat, catalase; MS, malate synthase.



As mentioned before, cells of per3 are still able to grow on ethanol in the presence of ethylamine as the sole nitrogen source. On these compounds the peroxisomal enzymes amine oxidase, catalase, and malate synthase are induced, whereas synthesis of the enzymes of the methanol metabolism is fully repressed. After fractionation of these cells, catalase and malate synthase were mainly present in the soluble fraction, whereas the bulk of the amine oxidase protein was sedimentable (Fig. 3B). Also Per1p, which contains both a PTS1 and PTS2, was predominantly present in the organellar pellet (data not shown; 12). After sucrose gradient density centrifugation of the organellar pellet, amine oxidase sedimented at a density of 52% (w/w) sucrose, which is typical for peroxisomes of WT cells(23) . A minor protein band recognized by catalase antibodies was observed in the peroxisomal peak fraction as well; however, malate synthase protein was not detectable in this fraction (Fig. 3B).

Subsequent (immuno)cytochemical experiments confirmed that amine oxidase protein was solely located inside the small peroxisomes of per3 cells (Fig. 4, A and B). Similar results were obtained for thiolase (Fig. 4D, inset); a heterologous PTS2 protein, namely watermelon gMDH, which is known to be imported in WT H. polymorpha peroxisomes, is also imported in the small peroxisomes of per3 cells. In contrast, PTS1 proteins (alcohol oxidase, catalase (not shown), and dihydroxyacetone synthase) were solely found in the cytosol together with malate synthase (Fig. 4, D-F). Taken together, we conclude that PTS1 proteins are not imported in per3 cells. The small amount of sedimentable catalase in sucrose gradients most probably results from aggregation or association of the protein with membrane vesicles.


Figure 4: Cytochemically, amine oxidase is solely present in the small peroxisomes (arrow) of glucose-choline grown cells (A). Typically, the membranous protrusions do not or only very slightly stain (inset). Immunocytochemically, using specific antibodies against amine oxidase, the protein is also found confined to the peroxisomal structures (B). A similar location is observed for watermelon gMDH in methanol-induced cells (C) and homologous thiolase (inset, D) in oleic-acid induced per3. Using specific antibodies against dihydroxyacetone synthase labeling was found in the cytosol (D). Also alcohol oxidase activity (E) and protein (F) were not found in the peroxisomal structures, but instead were located in the cytosol and nucleus. Due to the high expression rates in glucose/choline cells, the alcohol oxidase protein is present in large crystalloids (E and F) in which dihydroxyacetone synthase is also present (D). N, nucleus.



Per3p Is Located in the Cytosol and the Peroxisomal Matrix

Antibodies were raised against a Per3p-maltose-binding protein which was synthesized in E. coli. Using affinity purified antiserum (-Per3p), a single dominant protein band of approximately 70 kDa was found in crude extracts of H. polymorpha cells, overexpressing Per3p (PPER3; see below). In crude extracts of WT cells, grown on various carbon and nitrogen sources, Per3p was generally not detectable by Western blotting, indicating that the levels of Per3p are generally very low. However, in subcellular fractions from methanol grown cells (organellar pellet and peroxisomal peak fraction obtained after sucrose density centrifugation), a single protein band of approximately 70 kDa was clearly visualized on Western blots decorated with -Per3p (Fig. 5). A minor band of Per3p was found in blots prepared from the cytosolic fraction of these cells obtained after differential centrifugation of cell homogenates.


Figure 5: Subcellular localization of Per3p using Western blots decorated with affinity purified Per3p antibodies. In crude extracts of cells which overexpressed PER3, a distinct protein band with the apparent molecular mass of 70 kDa was detected (lane 1). This band was absent in crude extracts of methanol-induced per3 cells (lane 2). In crude extracts of methanol-grown WT cells (lane 3), this band was generally not detectable, although occasionally degradation products may be present (lane 3). Upon cell fractionation of methanol-grown WT cells, a dominant Per3p band was present in the organellar pellet (lane 4) and, less dominant, in the cytosolic fraction (lane 5). Per3p was clearly detectable in the peroxisomal peak fraction (lane 6), obtained after sucrose gradient centrifugation of the organellar pellet. In the mitochondrial peak fraction (lane 7), Per3p was absent. Upon carbonate treatment of peroxisomal peak fractions, the membrane pellet (lane 8) and soluble fraction (lane 9) were subjected to Western blotting; Per3p was only present in the supernatant (lane 9).



After carbonate treatment of peroxisomal peak fractions, Per3p was clearly present in the soluble fractions, suggesting that Per3p is not an integral membrane protein.

Immunocytochemical experiments, using -Per3p on ultrathin sections of WT H. polymorpha, revealed a dual location of Per3p in both the cytosol and the peroxisomal matrix. The peroxisomal labeling was found both on the alcohol oxidase crystalloids but also frequently at the periphery of the organelle (Fig. 6, A and C). The latter distribution is similar to the labeling pattern obtained for catalase, which is shown to be mainly located in the small zone between the alcohol oxidase crystalloid and the peroxisomal membrane(36) . In peroxisome-deficient mutants (per1; 12) labeling was solely found in the cytosol, in part associated with the cytosolic alcohol oxidase crystalloid (Fig. 6B). This suggests that Per3p is not membrane-bound because it resembles the behavior of other soluble matrix proteins such as amine oxidase and dihydroxyacetone synthase, which are predominantly found in association with alcohol oxidase crystalloids in peroxisome-deficient mutant cells(32) . Immunofluorescence experiments fully confirmed the above location of Per3p (Fig. 7).


Figure 6: A and C show the characteristic labeling patterns when specific antibodies against Per3p are used in the immunocytochemical experiments. Labeling is found both in peroxisomes and in the cytosol. Typically, peroxisomal labeling is found in the zone between the alcohol oxidase crystalloid and the peroxisomal membrane of partly crystalline organelles in batch cultured cells. In fully crystalline organelles from methanol-limited cells, the labeling is also found randomly over the matrix (A). In per1 disruption cells, which completely lack peroxisomal structures, the labeling is predominantly associated with the alcohol oxidase crystalloids (B). A similar labeling pattern (peroxisomal and cytosolic) is seen in ethanol-ethylamine grown cells, which only contain few small peroxisomes (D). After overexpression of Per3p (P-PER3), the labeling intensity of peroxisomes is not significantly enhanced, but more label is seen in the cytosol (E). In such cells alcohol oxidase protein is still confined to the peroxisomal matrix (F, anti-alcohol oxidase). N, nucleus.




Figure 7: Immunofluorescence. A and C, control labeling of amine oxidase in ethanol-ethylamine grown cells, showing the expected punctuate structures of the few peroxisomes present in these cells. A, phase contrast; C, immunofluorescence. B and D, phase contrast (B) and immunofluorescence (D) image of methanol-grown WT cells, containing cubic peroxisomes, using affinity purified anti-Per3p (compare C). E, labeling of peroxisomes in ethanol-ethylamine grown cells using affinity purified anti-Per3p; the cytosol shows slight fluorescence.



Overexpression of PER3 Does Not Affect the Import of Peroxisomal Matrix Proteins

Transformants, which expressed PER3 under control of the alcohol oxidase promoter (P), normally grew on methanol. In crude extracts, prepared from these cells, Per3p was readily detectable by Western blotting (Fig. 5), indicating that PER3 overexpression had occurred.

Immunocytochemical experiments revealed that also in these cells Per3p was both in peroxisomes and in the cytosol (Fig. 6E). Compared to WT control cells, the labeling intensity of the peroxisomal matrix was not significantly enhanced. The increased levels of Per3p also had no clearcut effect on the overall peroxisomal morphology and on the localization of typical PTS1 proteins; similar to WT cells, alcohol oxidase (Fig. 6F) and dihydroxyacetone synthase protein (not shown) were exclusively present in the matrix of the organelles present in these cells.


DISCUSSION

Per3p Is Dispensable for Sorting of PTS2 Proteins

The existence of different PTS-dependent pathways for peroxisomal protein import in yeasts was already predicted from the analysis of various import deficient mutants in which the import of specific subsets of peroxisomal proteins was fully prevented(7, 8, 9) . As such, these mutants resembled the phenotypes of six out of nine complementation groups of human Zellweger syndrome fibroblasts(37, 38) .

In this paper, we show that in cells of a H. polymorpha per3 disruption mutant (per3) PTS2 proteins are normally sorted to peroxisomes. These results indicate that peroxisomes of per3 cells contain a fully functional protein translocation machinery, thus independent of Per3p. PTS1 proteins however remained in the cytosol. Furthermore, since also heterologous PTS2 proteins are correctly imported, this PTS2 import pathway is, as the PTS1 import pathway, conserved to a certain extent.

Per3p Shows Similarity to P. pastoris Pas8p and S. cerevisiae Pas10p (the PTS1 or SKL receptor)

H. polymorpha Per3p shows significant similarity to Pas8p of P. pastoris and Pas10p of S. cerevisiae. Like the H. polymorpha per3 mutants, the P. pastoris pas8 and S. cerevisiae pas10 mutants are specifically defective in import of PTS1 proteins. The regions of strongest identity are found in the C-terminal parts, in which the TPR motif is located(31) . This motif is thought to play a role in protein-protein interactions; nevertheless, a precise biochemical function has not yet been assigned to it(39) . Most members of the TPR protein family play a role in mitosis or in regulation of the cell cycle; on the other hand, MOM72/MAS70, a mitochondrial protein receptor, also belongs to this family(40) . The receptor function of S. cerevisiae Pas10p is in fact confirmed in recent studies by Brocard et al.(41) , who showed that Pas10p interacts in vivo with the PTS1 and that the TPR motif is essential for this interaction.

The cytosolic PTS1 proteins in H. polymorpha per3 cells are correctly assembled and enzymatically active; this suggests that the putative peroxisomal factors(42) , involved in the assembly processes, are also not imported and functional in the cytosol. Remarkably, malate synthase which lacks a conserved PTS1 or PTS2 signal, also remained in the cytosol of per3 cells. A likely explanation for this result is that the yet unknown targeting signal of malate synthase is dependent on a PTS1 protein to facilitate import. A comparable explanation was presented by Kunau and co-workers (7) to account for the import of acyl-CoA oxidase and catalase A, which both have internal targeting signals, in S. cerevisiae pas7 cells.

At present, the subcellular location of the ``SKL receptor'' is still a matter of debate and may even be species-dependent. In P. pastoris Pas8p was mainly associated with peroxisomes (8) , whereas in S. cerevisiae Pas10p was predominantly found in the cytosol.()Our data, however, clearly demonstrated that in H. polymorpha the PER3 gene protein is located in each of these compartments and is not an integral component of the peroxisomal membrane.

What Is the Function of Per3p?

The easiest explanation, building on earlier results obtained with S. cerevisiae Pas10p and P. pastoris Pas8p, is that Per3p acts as a receptor, which specifically binds the PTS1 of peroxisomal precursors and shuttles these polypeptides from the cytosol to the organellar matrix. The fact that a major portion of Per3p is detected in peroxisomes may indicate that the release of Per3p from the organelles probably is rather slow compared to the other steps of the cycle.

An analogous situation may exist for PTS2 proteins. As indicated above, Kunau and co-workers (7) showed that in bakers' yeast Pas7p is specifically involved in the import of the PTS2 protein thiolase. Although ultrastructural methods failed to demonstrate myc-tagged Pas7p in the peroxisomal matrix, biochemical data pointed to a minor portion of myc-Pas7p being bound to peroxisomes. This result was strengthened by the finding that in a thiolase-deficient strain all Pas7p was invariably on the top of the gradient and thus fully soluble. Therefore, the association of Pas7p with peroxisomes is possibly dependent on its protein substrate, thiolase; probably, the EM technique was insufficiently sensitive to detect these low amounts of myc-tagged Pas7p in the organellar matrix.

If this assumption is correct, this implies that both the PTS1 and PTS2 import machineries may display the same basic mechanisms in that a cytosolic receptor (Per3p and eventually also P. pastoris Pas8p and S. cerevisiae Pas10p for PTS1 proteins and S. cerevisiae Pas7p for PTS2 proteins) is essential to recognize the protein to be imported and to direct it to the matrix of the target organelle.

How are these proteins imported? Taken together, the available data on in vivo and (semi) in vitro import studies are far from conclusive. In vivo the capacity of individual peroxisomes to incorporate newly synthesized proteins is only temporal (43) , a property which is not evident from import studies using semipermeabilized mammalian cells or protein microinjection(37) . Moreover, recently several examples have been described in which peroxisomal protein import is not restricted to proteins having an unfolded conformation(44, 45) . In addition, complex proteins, as for instance octameric alcohol oxidase or human serum albumin, to which SKL-containing peptides were chemically cross-linked(37) , were succesfully directed to peroxisomes after microinjection in mammalian cells. One could argue that import of the above proteins is preceded by their disassembly and unfolding at some point prior to the import; however, in particular in the case of octameric alcohol oxidase this is very unlikely(46) . Therefore, import of preassembled proteins into peroxisomes may indeed occur. From these results the picture emerges that peroxisomal import may be highly different from the basic principles underlying import in other organelles like mitochondria and chloroplasts. McNew and Goodman (45) discussed several interesting alternatives ranging from static to dynamic pores and membrane internalization processes. To elucidate the exact mechanisms of peroxisomal protein import is one of the main future challenges. There is little doubt that several proteins are involved in this process. For H. polymorpha Per3p genetic studies predict interactions with the gene products of PER4, PER5 (both members of the AAA family of ATPases and homologues of PAS1 and PAS8 of S. cerevisiae), and PER9 (the S. cerevisiae PAS3 homologue)(15).()

Based on the amino acid sequence similarity, we expect H. polymorpha Per3p to represent the functional homologue of P. pastoris Pas8p and S. cerevisiae Pas10p. However, the P. pastoris PAS8 gene could not functionally complement the H.polymorpha per3 strain although Pas8p was normally synthesized; immunocytochemistry revealed that the PAS8 gene product was largely cytosolic in H. polymorpha (data not shown).

Both P. pastoris Pas8p and H. polymorpha Per3p do not contain a conserved PTS1 or PTS2. Therefore, it is unclear how these proteins enter the organelle; one possibility includes that they are not imported in their free form, but that import is dependent on binding to their protein substrates. In line with this, the failure of the P. pastoris PAS8 gene to complement the H. polymorpha per3 mutant may be due to the fact that minor differences exist between the import machineries of both methylotrophic yeasts preventing the import, and thus the shuttling function, of Pas8p in H. polymorpha.

Concluding Remark

With the cloning and characterization of the H. polymorpha PER3 gene, three genes involved in peroxisome protein import have been isolated from three different yeast species. Although likely, it is not yet fully clear whether the protein products of these genes are functional homologues of each other and fulfill the same biochemical function. However, the availability of the deduced amino acid sequences of these proteins allows initiation of mutational studies in regions of interest to elucidate their function in detail.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U26678[GenBank® Link].

§
To whom correspondence should be addressed: Dept. for Microbiology, Biological Centre, Kerklaan 30, 9751 NN Haren, The Netherlands. Tel.: 31-50-632176; Fax: 31-50-635205; IJVDKLEI{at}biol.rug.nl

Supported by the Foundation for Fundamental Biological Research which is subsidized by the Netherlands Organization for the Advancement of Pure Research (SLW).

**
Supported by the Netherlands Technology Foundation (STW) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (SLW).

§§
Supported by National Science Foundation Grant MB-9118062 and National Institutes of Health Grant DK-43698.

The abbreviations used are: PTS, peroxisomal targeting signals; WT, wild type; kb, kilobase(s); PBS, phosphate-buffered saline.

Y. Elgersma and H. F. Tabak, personal communication.

J. A. K. W. Kiel, R. E. Hilbrands, and S. Rasmussen, unpublished results.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. S. Subramani (La Jolla) for providing the P. pastoris PAS8 gene and -Pas8p antibodies and Dr. W. H. Kunau (Bochum, Germany) for -thiolase antibodies. We thank Klaas-Nico Faber and Jan Kiel for expert advice in the molecular genetic part of the work. The skillfull assistance of Meis van der Heyden, Ineke Keizer-Gunnink, Klaas Sjollema, and Jan Zagers is gratefully acknowledged.


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