(Received for publication, September 23, 1994; and in revised form, November 2, 1994)
From the
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.
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
HO
forming oxidase and catalase to decompose
the H
O
(de Duve and Baudhuin, 1966). Depending
on the cell type or tissue, peroxisomes have been shown to be involved
in the
-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) ()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.
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).
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 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.
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 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).
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, 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 -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.
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).
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.
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.
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).
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16653[GenBank].