©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Yarrowia lipolytica Gene PAY2 Encodes a 42-kDa Peroxisomal Integral Membrane Protein Essential for Matrix Protein Import and Peroxisome Enlargement but Not for Peroxisome Membrane Proliferation (*)

(Received for publication, September 23, 1994; and in revised form, November 2, 1994)

Gary A. Eitzen (§) John D. Aitchison (¶) Rachel K. Szilard (**) Marten Veenhuis (1) William M. Nuttley (§§) Richard A. Rachubinski (¶¶)

From the Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada and theLaboratory for Electron Microscopy, University of Groningen, 9750 AA Haren, The Netherlands

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

PAY genes are required for peroxisome assembly in the yeast Yarrowia lipolytica. Here we show that a mutant strain, pay2, is disrupted for the import of proteins targeted by either peroxisomal targeting signal-1 or -2. Electron microscopy of pay2 cells revealed the presence of small peroxisomal ``ghosts,'' similar to the vesicular structures found in fibroblasts of patients with the human peroxisome assembly disorder, Zellweger syndrome. Functional complementation of pay2 with a plasmid library of Y. lipolytica genomic DNA identified a gene, PAY2, that restores growth of pay2 on oleic acid, import of catalase and multifunctional enzyme into peroxisomes, and formation of wild type peroxisomes. The PAY2 gene encodes Pay2p, a hydrophobic polypeptide of 404 amino acids. An antibody raised against Pay2p recognizes a polypeptide of 42-kDa whose synthesis is induced by growth of Y. lipolytica on oleic acid. Pay2p is a peroxisomal integral membrane protein, as it localizes to carbonate-stripped peroxisomal membranes. Pay2p shows no identity to any known protein. Our results suggest that Pay2p is essential for the activity of the peroxisomal import machinery but does not affect the initial steps of peroxisomal membrane proliferation.


INTRODUCTION

Peroxisomes are members of the microbody family of organelles, along with the glyoxysomes of plants and the glycosomes of trypanosomes. They are delimited by a single unit membrane and vary in size from 0.2 to 1.0 µm in diameter (for reviews, see Lazarow and Fujiki(1985) and Subramani(1993)). Peroxisomes contain at least one H(2)O(2) forming oxidase and catalase to decompose the H(2)O(2) (de Duve and Baudhuin, 1966). Depending on the cell type or tissue, peroxisomes have been shown to be involved in the beta-oxidation of long-chain fatty acids (Lazarow and de Duve, 1976), bile acid synthesis (Krisans et al., 1985), plasmalogen synthesis (Hajra and Bishop, 1982), cholesterol metabolism (Thompson et al., 1987), and methanol oxidation (van der Klei et al., 1991).

Peroxisomes are required for normal human development and physiology, as shown by the lethality of human genetic disorders like Zellweger syndrome in which peroxisome assembly is disrupted. Zellweger syndrome cells contain peroxisomal ``ghosts,'' vesicular structures largely or completely devoid of matrix proteins (Santos et al., 1988a, 1988b). It has been proposed that Zellweger syndrome arises from a failure to translocate proteins into peroxisomes (Walton et al., 1992; Wendland and Subramani, 1993), causing these proteins to be mislocalized to the cytoplasm where they cannot function correctly and are often rapidly degraded. There are at least nine different complementation groups in Zellweger syndrome, suggesting that a number of genes are involved in normal human peroxisome assembly (Yajima et al., 1992; Shimozawa et al., 1992). Two genes have been identified so far. One encodes a 35-kDa peroxisomal integral membrane protein called peroxisome assembly factor-1 (Shimozawa et al., 1992), while the second encodes a 70-kDa peroxisomal integral membrane protein called PMP-70 (Gärtner et al., 1992).

All peroxisomal proteins are encoded in the nucleus and synthesized on polysomes free in the cytoplasm. Therefore, proteins are post-translationally translocated into the peroxisomal matrix or membrane. Two targeting signals have been identified in the transport of matrix proteins. Peroxisomal targeting signal-1 (PTS-1) (^1)is a tripeptide motif found at the carboxyl termini of many peroxisomal matrix proteins in organisms from yeast to humans (Gould et al., 1989, 1990; Aitchison et al., 1991). PTS-1 motifs are identical to or conserved variants of the prototypical Ser-Lys-Leu targeting signal of firefly luciferase (Gould et al., 1987, 1988). PTS-2 motifs are amino-terminal signals first identified in rat peroxisomal thiolase (Swinkels et al., 1991; Osumi et al., 1991) and which may or may not be proteolytically cleaved upon import (Glover et al., 1994). To date there is only limited information on signals that target proteins to the peroxisomal membrane (McCammon et al., 1994).

Yeast represent an excellent experimental system by which the genes controlling the cellular events leading to peroxisome assembly can be identified. Peroxisome assembly mutants have been isolated in Saccharomyces cerevisiae (Erdmann et al., 1989; van der Leij et al., 1992; Zhang et al., 1993), Hansenula polymormpha (Cregg et al., 1990), Pichia pastoris (Liu et al., 1992; Gould et al., 1992), and Yarrowia lipolytica (Nuttley et al., 1993). Functional complementation of some of these mutants have identified genes encoding a varied set of proteins involved in peroxisome biogenesis including putative ATPases (Erdmann et al., 1991; Spong and Subramani, 1993; Nuttley et al., 1994), tetratricopeptide-repeat proteins that may be part of the import machinery for PTS-1-targeted proteins (McCollum et al., 1993; van der Leij et al., 1993), a peroxisomal integral membrane protein (Höhfeld et al., 1991), and proteins related to ubiquitin-conjugating enzymes (Wiebel and Kunau, 1992; Crane et al., 1994).

Herein we report the detailed morphological and biochemical characterization of a peroxisome assembly mutant strain of Y. lipolytica, pay2. pay2 fails to assemble normal peroxisomes; however, it does form vesicular structures similar to the peroxisomal ghosts seen in Zellweger cells. pay2 cells fail to import proteins containing either PTS-1 or PTS-2 motifs. Functional complementation of the pay2 mutant yielded the PAY2 gene, which encodes a hydrophobic protein of 404 amino acids, Pay2p. Pay2p is a peroxisomal integral membrane protein that is unrelated to any known protein.


MATERIALS AND METHODS

Strains and Media

The yeast strains used in this study are given in Table 1. Yeast were grown in complete (YEPD) or minimal (YNO, YND, and induction) media, as required. YEPD medium contained 1% yeast extract, 2% peptone, 2% glucose; YNO medium contained 0.67% yeast nitrogen base without amino acids, 0.05% (w/v) Tween 40, 0.1% (w/v) oleic acid; YND medium contained 0.67% yeast nitrogen base without amino acids, 2% glucose; induction medium contained 0.67% yeast nitrogen base without amino acids, 0.5% yeast extract, 0.5% peptone, 0.1% glucose, 0.1% (w/v) oleic acid, 0.5% (w/v) Tween 40 (Erdmann et al., 1989). Media were supplemented with uracil, leucine, lysine, and histidine each at 50 µgbulletml, as required. Growth was at 30 °C unless specified otherwise.



Isolation of the pay2 Mutant Strain

The pay2 mutant strain was isolated after mutagenesis of E122 cells with 1-methyl-3-nitro-1-nitrosoguanidine (Nuttley et al., 1993). The screening protocol included selection for the inability to use oleic acid as a carbon source, fractionation into 20kgP (primarily peroxisomes and mitochondria) and 20kgS (primarily cytosol) fractions of yeast cells (Aitchison et al., 1991), and electron microscopy (Waterham et al., 1992; Nuttley et al., 1994). Mutants were characterized by standard genetic techniques for Y. lipolytica (Gaillardin et al., 1973).

Marker Enzyme Analyses

The following marker enzyme activities were measured: peroxisomes, catalase (Baudhuin et al., 1964), beta-hydroxyacyl-CoA dehydrogenase (Osumi and Hashimoto, 1979; mitochondria, cytochrome c oxidase (Cooperstein and Lazarow, 1951).

Cloning and Characterization of the PAY2 Gene

The PAY2 gene was isolated by functional complementation of the pay2 strain using a genomic DNA library of Y. lipolytica contained in the Escherichia coli shuttle vector pINA445 (Nuttley et al., 1993). Plasmids were introduced into cells by electroporation (Nuttley et al., 1993). Leu transformants were screened on YNO-agar plates for their ability to use oleic acid as the sole carbon source. Complementing plasmids were recovered by transformation of E. coli. Standard recombinant DNA methodology including enzymatic modification of DNA, DNA fragment purification, and plasmid isolation, was performed essentially as described in Ausubel et al. (1989).

DNA Sequencing

Various restriction endonuclease fragments of the PAY2 gene were cloned into the vectors pGEM-5Zf(+) and pGEM-7Zf(+) (Promega, Madison, WI) for dideoxynucleotide sequencing of both strands from double-stranded templates (Sanger et al., 1977; Zhang et al., 1988). The deduced Pay2p sequence was compared to other known protein sequences to determine similarities using the GENINFO(R) BLAST Network Service (Blaster) of the National Center for Biotechnology Information.

Integrative Disruption of the PAY2 Gene

Integrative disruption of the PAY2 gene was done with the LEU2 gene of Y. lipolytica. The plasmid pINA445 was cleaved into two fragments with BglII. The ends were made blunt with T4 DNA polymerase. The fragment containing the LEU2 gene was isolated by electroelution after agarose gel electrophoresis. The isolated DNA fragment was cleaved with Eco47III. The Eco47III/blunt BglII fragment containing the LEU2 gene was inserted into the PAY2 gene cleaved with EagI and HindIII made blunt with the Klenow fragment of DNA polymerase I. This construction replaced 222 base pairs (bp) in the coding region of the PAY2 gene open reading frame with an approximately 2.1-kbp fragment. A construct with the open reading frame of the LEU2 gene opposite to that of the PAY2 gene was selected. This construct was digested with SalI and BamHI to liberate the LEU2 gene flanked by 697 and 1224 bp of the PAY2 gene at its 5` and 3` ends, respectively. This linear molecule was used to transform Y. lipolytica to leucine prototrophy. Leu transformants were screened for the ole phenotype and mated to 22301-3. Diploids were sporulated and random spore analysis performed. The segregation of the leu and ole phenotypes was analyzed by replica plating. pay2::LEU2 segregants were mated to pay2 and the resultant diploids checked for complementation.

Antisera

To produce antibodies to Pay2p, a 732-bp fragment of the PAY2 gene open reading frame encoding amino acids 76-318 of Pay2p was excised with StyI, made blunt with T4 DNA polymerase, and inserted into the XmnI site of pMAL-c2 (New England Biolabs) in-frame and downstream of the open reading frame encoding the maltose-binding protein. A lysate of E. coli synthesizing the maltose-binding protein-Pay2p fusion was prepared essentially as described by Ausubel et al.(1989), and the fusion protein was further purified by SDS-PAGE (Laemmli, 1970; Fujiki et al., 1984) on a 10% preparative gel. The fusion protein was electroeluted into dialysis tubing, dialyzed against several changes of 50 mM ammonium bicarbonate, lyophilized, and dissolved in a minimal amount of distilled, deionized water. Antibodies were raised in guinea pigs as described previously (Nuttley et al., 1994). Anti-S. cerevisiae peroxisomal thiolase was a gift of Dr. W.-H Kunau, Ruhr University.

The specificities of antisera were determined by Western blotting (Burnette, 1981) of yeast cell lysates (Needleman and Tzagoloff, 1975; Nuttley et al., 1993). Antigen-antibody complexes were detected by enhanced chemiluminescence (Amersham) or I-protein A (DuPont NEN).

Isolation of Peroxisomes and Peroxisomal Membranes

Fractions enriched for peroxisomes were isolated from Y. lipolytica E122 grown 12 h in YPBO medium (0.3% yeast extract, 0.5% peptone, 0.5% K(2)HPO(4), 0.5% KH(2)PO(4), 1.0% Brij-35, 1.0% (w/v) oleic acid) by differential centrifugation followed by isopycnic centrifugation on sucrose gradients (Kamiryo et al., 1982). Peroxisomes were divided into a pellet fraction containing membranes and a soluble fraction containing matrix by sodium carbonate extraction (Fujiki et al., 1982).

Isolation of Nucleic Acids and Northern Blot Analysis

Y. lipolytica E122 grown overnight in YEPD was pelleted by centrifugation, washed in sterile water, and transferred to YPBO. Samples were removed at various times after transfer to YPBO. Whole cell lysates were made by disruption of cells with glass beads. Isolation of nucleic acids and Northern blot analysis were performed essentially as described by Ausubel et al. (1989).

Miscellaneous Procedures

Total protein content was determined as described by Bradford(1976). Densitometry was performed using a LKB Ultroscan XL laser densitometer (LKB Instruments, Bromma, Sweden).


RESULTS

Pay2 Cells Can Proliferate Peroxisome-like Structures but Are Deficient for Peroxisome Growth

The Y. lipolytica mutant strain pay2 is incapable of growth on oleic acid (Fig. 1). pay2 is a mutant of peroxisome assembly, as it aberrantly localizes peroxisomal marker enzymes to the 20kgS cytosolic fraction (Table 2). Analysis of the parental E122 and mutant pay2 strains by electron microscopy supports the classification of the pay2 strain as being compromised in peroxisome assembly. E122 cells grown in oleic acid medium show peroxisomes scattered throughout the cytoplasm (Fig. 2, C and D). In contrast, pay2 cells grown in oleic acid show small peroxisome-like vesicular structures (Fig. 2, A and B), reminiscent of the peroxisomal ghosts seen in Zellweger cells (Santos et al., 1988a, 1988b). These peroxisome-like structures appear less granular than wild type peroxisomes and have an average diameter of 0.2 µm, in contrast to the average diameter of 0.7 µm of peroxisomes in the parental strain E122. Therefore, pay2 cells appear to be able to proliferate peroxisome-like structures but are deficient for subsequent growth of the organelle.


Figure 1: Growth of various Y. lipolytica strains on oleic acid medium. Appearance of the complemented strain PAY2 in comparison to the parental strain E122, the mutant strain pay2, the gene disruption strain P2-KO, the diploid D2-22 (P2-KO X 22301-3), and a second parental strain 22301-3 (the plate was not supplemented to satisfy the auxotrophic requirements of this strain). Growth was for 4 days on YNO-agar.






Figure 2: Ultrastructure of the pay2 mutant (Panels A and B) and parental E122 (Panels C and D) strains. Cells were grown to saturation in YEPD medium, diluted 1:4 into YNO medium, and grown for an additional 24 h. The cells were then fixed in KMnO(4) and processed for electron microscopy. Arrows point to some of the small vesicular structures reminiscent of the peroxisomal ghosts of Zellweger fibroblasts. P, peroxisomes; N, nucleus; V, vacuole. Bar = 1 µm.



pay2 Cells Mislocalize a PTS-2-containing Protein to the Cytoplasm

We have previously shown that pay2 cells mislocalize some proteins containing PTS-1 motifs to the cytoplasm (Nuttley et al., 1993). pay2 cells also mislocalize a PTS-2-containing protein, i.e. thiolase, to the cytoplasm (Fig. 3). A Western blot of the 20kgP (primarily peroxisomes and mitochondria) and 20kgS (primarily cytosol; Aitchison et al.(1991)) fractions of the parental E122 strain showed a single polypeptide band of molecular weight approximately 43,000 exclusively in the 20kgP fraction when probed with anti-thiolase serum (Fig. 3, E122, laneP). In pay2 cells, thiolase was not localized to the 20kgP (Fig. 3, pay2, laneP) but mislocalized to the 20kgS (Fig. 3, pay2, laneS). The majority of the thiolase in the 20kgS was the larger precursor form of the protein (Nuttley et al., 1994), although some mature form of the protein and apparent intermediates of thiolase precursor cleavage were also seen. The origins of these intermediate forms are unknown.


Figure 3: Peroxisomal thiolase is localized to the 20kgS cytosolic fraction of the mutant pay2 strain. The equivalent cellular fractions of the supernatant (S) and pellet (P) fractions from a 20,000 times g centrifugation of postnuclear supernatants were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antiserum to S. cerevisiae peroxisomal thiolase. The numbers at left indicate the migrations of molecular mass standards (in kDa).



Isolation of the PAY2 Gene

Transformation of the pay2 strain with a plasmid library containing Y. lipolytica genomic DNA yielded 4 transformants capable of restored growth on oleic acid. Four independent recombinant plasmids, designated pO2-A to pO2-D, were rescued into E. coli. Restriction analysis followed by subcloning and transformation of the pay2 mutant showed the complementing activity of the inserts was localized to a common minimal 2.2-kbp segment (data not shown). A transformant, hereafter called PAY2 (Fig. 1), harboring the plasmid pO2-2.2 containing the common 2.2-kbp segment, was used for further study.

The putative PAY2 gene was used for a gene disruption experiment with the Y. lipolyticaLEU2 gene, as described under ``Materials and Methods.'' A leu/ole transformant, designated P2-KO, was isolated (Fig. 1). Integration of the LEU2 gene into the PAY2 locus was confirmed by Southern blotting (data not shown). The recessive nature of the ole phenotype was shown by the ability of the P2-KO X 22301-3 diploid to grow on oleic acid (Fig. 1, D2-22). Sporulation of the diploid D2-22 showed cosegregation of the ole and leu phenotypes. When an ole, MATB isolate (P2KO-3) from the sporulation of D2-22 was back-crossed to the original pay2 strain, the resultant diploid (D3-301) was unable to grow on oleic acid, thereby confirming that the authentic PAY2 gene had been cloned.

Electron microscopic examination of cells of the PAY2 transformant grown in oleic acid medium showed peroxisomes like those found in the parental strain E122 (Fig. 4, C and D). In contrast, the P2-KO strain (Fig. 4, A and B) showed vesicular peroxisomal ghosts like those seen in the original pay2 mutant.


Figure 4: Ultrastructure of the P2-KO mutant (Panels A and B) and the PAY2 transformant (Panels C and D). Cells were induced in YNO medium, fixed with KMnO(4), and processed for electron microscopy. The electron micrographs show the morphology of the P2-KO mutant to be similar to the pay2 strain. Arrows point to some of the small vesicular structures reminiscent of peroxisomal ghosts. P, peroxisomes; N, nucleus; V, vacuole. Bar = 1 µm.



Transformation of pay2 with pO2-2.2 to yield PAY2 also restored the correct localization of peroxisomal marker enzyme activities. In the parental strain E122, approximately 50% of the catalase activity and 60% of the beta-hydroxyacyl-CoA dehydrogenase activity were found in the 20kgP fraction upon subcellular fractionation (Table 2), reflecting the peroxisomal location of these enzymes. The activities of these enzymes recovered in the 20kgS fraction were due, at least in part, to leakage from peroxisomes broken during the fractionation procedure (Aitchison et al., 1991). In the pay2 mutant strain, as well as in the disrupted strain P2-KO, less than 4% of catalase activity and less than 10% of dehydrogenase activity were recovered in the 20kgP. Transformation of pay2 with pO2-2.2 corrected this defect, resulting in recoveries of catalase and dehydrogenase activities in the 20kgP fraction at levels similar to those found in the E122 parental strain. The preferential localization of the mitochondrial marker cytochrome c oxidase was not affected by the pay2 mutation, as comparable levels of cytochrome c oxidase activity were found in the 20kgP fractions of E122, pay2, PAY2, and P4-KO strains.

Nucleotide Sequence of the PAY2 Gene and the Deduced Amino Acid Sequence of Pay2p

Sequencing of the approximately 2.2-kbp insert of the complementing plasmid pO2-2.2 revealed an open reading frame encoding a protein of 404 amino acids and having a predicted molecular weight of 44,913 (Fig. 5). This predicted molecular weight is in good agreement with the relative molecular weight determined by SDS-PAGE and Western blot analysis (see Fig. 9). Three potential initiation codons are found within the first 10 amino acids of the protein encoded by the largest open reading frame, with two of them, the first and the third, conforming to the consensus sequence for translation initiation in yeast, with a conserved A at position -3 and a conserved C at position +5 relative to the A of the initiation codon (Cigan and Donahue, 1987). Which codon is the actual initiation codon is not known at this time. A putative TATA element, TACAAATAA, is found between positions -117 and -109 of the first potential initiation codon.


Figure 5: Nucleotide sequence and deduced amino acid sequence of the PAY2 gene. A presumptive consensus TATA sequence is shadowed. Two possible initiator methionines are double underlined. The two most likely membrane-spanning segments are highlighted in bold and underlined. A third potential membrane-spanning segment is highlighted in bold. Two additional segments that are potentially membrane-associated are underlined.




Figure 9: Pay2p is induced by growth of Y. lipolytica in oleic acid. E122 cells were grown for 16 h in glucose medium (YEPD) (lane a), diluted 1:5 in oleic acid medium (YPBO), and grown for 1 (lane b), 6 (lane c), and 12 (lane d) h in YPBO. The P2-KO (lane e) and pay2 (lane f) strains were grown for 16 h in YND, diluted 1:5 in YNO, and grown in YNO for 6 h. Equal amounts of protein from each sample were analyzed by SDS-PAGE, transferred to nitrocellulose, and probed with anti-Pay2p serum. The numbers at the left indicate the migrations of molecular mass standards (in kDa).



Analysis of the Pay2p Sequence

A search of protein data bases using the GENINFO(R) BLAST Network Service (Blaster) of the National Center for Biotechnology Information revealed no significant identity or homology of Pay2p with any known protein sequence. Hydropathy analysis (Fig. 6; Kyte and Doolittle(1982)) showed Pay2p to be hydrophobic overall and most likely a membrane protein. Based on algorithms predicting membrane-spanning regions in proteins (Eisenberg et al., 1984; Klein et al., 1985; Rao and Argos, 1986), Pay2p is predicted to contain two membrane-spanning alpha-helices located toward its amino terminus (Fig. 5, bold and underlined sequences). Pay2p is also predicted to contain a membrane-spanning or membrane-associated domain (Fig. 5, bold sequence) and two additional membrane-associated helices (Fig. 5, underlined sequences).


Figure 6: Hydropathy analysis of Pay2p. A hydropathy profile of the predicted amino acid sequence of Pay2p was calculated according to Kyte and Doolittle(1982) with a window size of 19 amino acids. The threshold hydrophobicity value of 25 is indicated by the solid horizontal line. The sequence highlighted in black contains the two most likely membrane-spanning domains. The sequence highlighted by cross-hatching also contains a putative membrane-spanning domain. The two sequences highlighted by vertical lines are most likely membrane-associated.



Pay2p Is a Peroxisomal Integral Membrane Protein

Since computer analysis predicted Pay2p to be a membrane protein and since the pay2 strain is deficient in peroxisome assembly, we wondered whether Pay2p was a peroxisomal membrane protein. Western blot analysis of subcellular fractions of oleic acid-grown E122 cells with anti-Pay2p serum (Fig. 7B) showed that Pay2p was preferentially localized to the fraction of peak peroxisomal catalase activity (Fig. 7A). Sodium carbonate extraction of this fraction (Fig. 7C, fraction 4) showed that Pay2p was exclusively found in the pellet, strongly indicative of Pay2p being an integral peroxisomal membrane protein.


Figure 7: Pay2p is a peroxisomal integral membrane protein. Panel A, marker enzyme analysis of a density gradient of the 20kgP of E122 cells grown in oleic acid medium. Fractions of equal volume were collected from the bottom of the tube. Panel B, anti-Pay2p was used to probe a Western blot of the fractions collected in Panel A. Panel C, fraction 4 was treated with sodium carbonate as described under ``Materials and Methods'' and then subjected to ultracentrifugation to yield a supernatant (S) of soluble proteins and a pellet (P) enriched for peroxisomal integral membrane proteins. Equal cellular fractions were separated on a SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with anti-Pay2p serum.



The Levels of Pay2p mRNA and Pay2p Are Induced by Growth On Oleic Acid

Growth of yeast on oleic acid leads to induced levels of mRNA coding for peroxisomal proteins and proteins involved in peroxisome assembly and to induced levels of the proteins themselves (Fujiki et al., 1986; Erdmann et al., 1991; Höhfeld et al., 1991; Nuttley et al., 1994). Growth of the parental strain E122 in medium containing glucose led to a low level of Pay2p mRNA (Fig. 8, left panel, lane 0). This low level of Pay2p mRNA was comparable to the low level of mRNA encoding 3-ketoacyl-CoA thiolase, a soluble peroxisomal beta-oxidation enzyme, found in the same cells (Fig. 8, right panel, lane 0). Shifting of the glucose-grown cells to medium containing oleic acid, followed by continued growth in this medium, brought about an increase in the levels of both Pay2p mRNA and thiolase mRNA. Four h after the shift to oleic acid medium, Pay2p mRNA levels were increased approximately 20-fold over the levels found in glucose-grown cells. Thereafter, Pay2p mRNA levels dropped and then stabilized starting at 6 h after the shift at levels approximately 4 times that found in glucose-grown cells. Thiolase mRNA was maximally induced (approximately 50-fold) 2 h after shifting cells to oleic acid medium, and this high level of induction was maintained even after 8 h growth in oleic acid medium.


Figure 8: Pay2p mRNA and peroxisomal thiolase mRNA are induced by growth of Y. lipolytica in oleic acid. Total RNA was isolated from strain E122 grown in glucose medium (YEPD; 0 HRS) and after transfer to oleic acid medium (YPBO) for different periods of times (numbers at the top of the figure indicate hours). 10 µg of RNA from each time point was analyzed on a formaldehyde-agarose gel and transferred to nitrocellulose. The blots were hybridized with radiolabeled probes specific for the PAY2 gene and the POT1 gene encoding S. cerevisiae peroxisomal thiolase. The numbersbetween the two panels represent the electrophoretic migrations of DNA markers (in kbp). Exposure was 5 days for the Pay2p mRNA and 15 h for the thiolase mRNA.



The levels of Pay2p itself were also increased by growth of E122 in medium containing oleic acid (Fig. 9). Pay2p was essentially undetectable in glucose-grown cells (compare the signal in lane e, glucose-grown E122, to the signal in lane f, P2-KO). Transfer of glucose-grown E122 cells to oleic acid medium followed by continued growth in this medium led to increased levels of Pay2p (lanes a-d), with maximum levels (10-fold induction) seen by 6 h (lane c).


DISCUSSION

The pay2 strain is a peroxisome assembly mutant that does not contain normal-looking peroxisomes but instead contains smaller vesicular structures reminiscent of the ``peroxisomal ghosts'' seen in fibroblasts of patients with Zellweger syndrome. Therefore, pay2 appears to be a mutant in which import of proteins into the peroxisome and peroxisome enlargement, but not proliferation of the peroxisomal membrane, are disrupted. It has been shown that peroxisomal membrane proliferation precedes protein import into peroxisomes (Veenhuis and Goodman, 1990; McCollum et al., 1993) and that the enlargement of peroxisomes is the result of protein import into initially much smaller peroxisomes (Godecke et al., 1989). In the pay2 (Fig. 2, C and D) and P2-KO (Fig. 4, A and B) strains, several small peroxisome-like ghosts can be seen. However, in the mutant strains, enlargement of peroxisomes is retarded after the initial proliferation of peroxisomes. Therefore, peroxisomal membrane proliferation can be genetically separated from protein import into peroxisomes. These morphological results, combined with the demonstration that the pay2 strain fails to import proteins targeted by either PTS-1 or PTS-2, suggest an association of Pay2p with the peroxisomal protein import machinery.

Pay2p represents the first peroxisomal protein integral membrane shown to be essential for the import of proteins into peroxisomes but not for peroxisomal membrane proliferation. Mutation of Pas3p, a peroxisomal integral membrane protein required for peroxisome assembly in S. cerevisiae, compromises both protein import into peroxisomes and peroxisomal membrane proliferation, resulting in a total absence of any peroxisomes or peroxisome-like structures (Höhfeld et al., 1991). Putative PTS-1 receptors have been identified in P. pastoris (Pas8p; McCollum et al.(1993)) and S. cerevisiae (Pas10p; van der Leij et al. (1993)); however, these proteins apparently have only a transient association with the peroxisomal membrane and are not integral to the membrane.

It is interesting to speculate that the initial events in targeting proteins with PTS-1, PTS-2, or other signals to peroxisomes are divergent with each signal being recognized by a separate receptor that can shuttle between the cytoplasm and the peroxisomal membrane. Such cytosolic factors have been predicted to exist for all organelles and would help in the targeting of proteins to specific organelles by preventing preproteins interacting with the incorrect membrane surface (Lithgow et al., 1993). At the peroxisomal membrane, these receptor-peroxisomal protein complexes would converge and interact with a single peroxisomal import machinery. We are in the process of conducting experiments aimed at determining whether Pay2p is a constituent of or interacts directly with this peroxisomal import apparatus.


FOOTNOTES

*
This work was funded in part by the Natural Sciences and Engineering Research Council of Canada. 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(TM)/EMBL Data Bank with accession number(s) U16653[GenBank].

§
Recipient of a Studentship from the Alberta Heritage Foundation for Medical Research.

Current address: Laboratory for Cell Biology, The Rockefeller University, New York, NY, 10021-6399.

**
Recipient of a Studentship from the Medical Research Council of Canada.

§§
Current address: Dept. of Biology, University of California at San Diego, La Jolla, CA 92093-0322.

¶¶
Medical Research Council of Canada Scientist. To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, University of Alberta, Medical Sciences Bldg., Edmonton, Alberta T6G 2H7, Canada. Tel.: 403-492-9868; Fax: 403-492-0450; rrachubi{at}anat.med.ualberta.ca.

(^1)
The abbreviations used are: PTS, peroxisomal targeting signal; 20kgP, 20,000 times g pellet (primarily peroxisomes and mitochondria); 20kgS, 20,000 times g supernatant (primarily cytosol); bp, base pair; kbp, kilobase pair; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. W.-H. Kunau for anti-S. cerevisiae thiolase serum and Dr. E. Schweizer for the Y. lipolytica thiolase clone, pS106.


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