Hansenula polymorpha: An attractive model organism for molecular studies of peroxisome biogenesis and function

In wild-type Hansenula polymorpha the proliferation of peroxisomes in induced by various unconventional carbon- and nitrogen sources. Highest induction levels, up to 80% of the cytoplasmic volume, are observed in cells grown in methanol-limited chemostat cultures. Based on our accumulated experience, we are now able to precisely adjust both the level of the peroxisome induction as well as their protein composition by specific adaptations in growth conditions. During the last few years a series of "peroxisome-deficient (per) mutants of H. polymorpha have been isolated and characterized. Phenotypically these mutants are characterized by the fact that they are not able to grow on methanol. Three mutant phenotypes were defined on the basis of morphological criteria, namely: (a) mutants completely lacking peroxisomes (Per-;13 complementation groups); (b) mutants containing few small peroxisomes which are partly impaired in the peroxisomal import of matrix proteins (Pim-; five complementation groups); and (c) mutants with aberrations in the peroxisomal substructure (Pss-; two complementation groups). In addition, several conditional Per-, Pim- and Pss- mutants have been obtained. In all cases the mutant phenotype was shown to be caused by a recessive mutation in one gene. However, we observed that different mutations in one gene may cause different morphological mutant phenotypes. A detailed genetic analysis revealed that several PER genes, essential for peroxisome biogenesis, are tightly linked and organized in a hierarchical fashion. The use of both constitual and conditional per mutants in current and future studies of the molecular mechanisms controlling peroxisome biogenesis and function is discussed.     

Cloning and characterization of PAS5: a gene required for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris

The biogenesis and maintenance of cellular organelles is of fundamental importance in all eukaryotic cells. One such organelle is the peroxisome. The establishment of a genetic system to study peroxisome biogenesis in the methylotrophic yeast Pichia pastoris has yielded many different complementation groups of peroxisomal assembly (pas) or peroxisome-deficient (per) mutants. Each appears to be deficient in functional peroxisomes. One of these mutants, pas5, has been characterized, complemented, and the gene sequenced. Ultrastructural studies show that normal peroxisomes are not present in pas5, but aberrant peroxisomal structures resembling "membranous ghosts" are frequently observed. The "peroxisome ghosts" appear to be induced and segregated to daughter cells normally. Biochemical fractionation analysis of organelles of the pas5 mutant reveals that peroxisomal matrix enzymes are induced normally but are found mostly in the cytosol. However, purification of peroxisome ghosts from the mutant shows that small amounts (< 5%) of matrix enzymes are imported. The PAS5 gene was cloned and found to encode a 127-kD protein, which contains a 200-amino acid-long region of homology with PAS1, NEM- sensitive factor (NSF), and other related ATPases. Weak homology to a yeast myosin was also observed. The gene is not essential for growth on glucose but is essential for growth on oleic acid and methanol. The role of PAS5 in peroxisome biogenesis is discussed.     

Role of the PAS1 gene of Pichia pastoris in peroxisome biogenesis

Several groups have reported the cloning and sequencing of genes involved in the biogenesis of yeast peroxisomes. Yeast strains bearing mutations in these genes are unable to grow on carbon sources whose metabolism requires peroxisomes, and these strains lack morphologically normal peroxisomes. We report the cloning of Pichia pastoris PAS1, the homologue (based on a high level of protein sequence similarity) of the Saccharomyces cerevisiae PAS1. We also describe the creation and characterization of P. pastoris pas1 strains. Electron microscopy on the P. pastoris pas1 cells revealed that they lack morphologically normal peroxisomes, and instead contain membrane-bound structures that appear to be small, mutant peroxisomes, or "peroxisome ghosts." These "ghosts" proliferated in response to induction on peroxisome-requiring carbon sources (oleic acid and methanol), and they were distributed to daughter cells. Biochemical analysis of cell lysates revealed that peroxisomal proteins are induced normally in pas1 cells. Peroxisome ghosts from pas1 cells were purified on sucrose gradients, and biochemical analysis showed that these ghosts, while lacking several peroxisomal proteins, did import varying amounts of several other peroxisomal proteins. The existence of detectable peroxisome ghosts in P. pastoris pas1 cells, and their ability to import some proteins, stands in contrast with the results reported by Erdmann et al. (1991) for the S. cerevisiae pas1 mutant, in which they were unable to detect peroxisome-like structures. We discuss the role of PAS1 in peroxisome biogenesis in light of the new information regarding peroxisome ghosts in pas1 cells.     

The transcarboxylase domain of pyruvate carboxylase is essential for assembly of the peroxisomal flavoenzyme alcohol oxidase

Pyruvate carboxylase (Pyc1p) has multiple functions in methylotrophic yeast species. Besides its function as an enzyme, Pyc1p is required for assembly of peroxisomal alcohol oxidase (AO). Hence, Pyc1p-deficient cells share aspartate auxotrophy (Asp-) with a defect in growth on methanol as sole carbon source (Mut-). To identify regions in Hansenula polymorpha Pyc1p that are required for the function of HpPyc1p in AO assembly, a series of random mutations was generated in the HpPYC1 gene by transposon mutagenesis. Upon introduction of 18 mutant genes into the H. polymorpha PYC1 deletion strain (pyc1), four different phenotypes were obtained, namely Asp- Mut-, Asp- Mut+, Asp+ Mut-, and Asp+ Mut+. One mutant showed an Asp+ Mut- phenotype. This mutant produced HpPyc1p containing a pentapeptide insertion in the region that links the conserved N-terminal biotin carboxylation domain (BC) with the central transcarboxylation (TC) domain. Three mutants that were Asp- Mut- contained insertions in the TC domain, suggesting that this domain is important for both functions of Pyc1p. Analysis of a series of constructed C-terminal and N-terminal truncated versions of HpPyc1p showed that the TC domain of Pyc1p, including the region linking this domain to the BC domain, is essential for AO assembly.     

Lipid composition of peroxisomes from the yeast Pichia pastoris grown on different carbon sources

Highly purified peroxisomes from the yeast Pichia pastoris grown on methanol or oleic acid, respectively, were used to characterize the lipid composition of this organelle. For this purpose, an isolation procedure had to be adapted which yielded highly purified P. pastoris peroxisomes. When peroxisome proliferation was induced by growth on methanol, alcohol oxidase was the predominant peroxisomal protein. Cultivation of P. pastoris on oleic acid led to induction of a family of peroxisomal enzymes catalyzing fatty acid beta-oxidation, whose most prominent members were identified by mass spectrometry. On either carbon source, phosphatidylcholine and phosphatidylethanolamine were the major peroxisomal phospholipids, and cardiolipin was present in peroxisomal membranes at a substantial amount, indicating that this phospholipid is a true peroxisomal component. Ergosterol was the most abundant sterol of P. pastoris peroxisomal membranes irrespective of the culture conditions. The fatty acid composition of whole cells and peroxisomes was highly affected by cultivation of P. pastoris on oleic acid. Under these conditions, oleic acid became the predominant fatty acid in phospholipids from total cell and peroxisomal extracts. Thus, oleic acid was not only utilized as an appropriate carbon source but also as a building block for complex membrane lipids. In summary, our data provide first insight into biochemical properties of P. pastoris peroxisomal membranes, which may become important for the biotechnological use of this yeast.     

Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris

We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX groups.     

The peroxisome biogenesis factors pex4p, pex22p, pex1p, and pex6p act in the terminal steps of peroxisomal matrix protein import

Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.     

Phosphorylation-dependent Pex11p and Fis1p interaction regulates peroxisome division

Peroxisome division is regulated by the conserved peroxin Pex11p. In Saccharomyces cerevisiae (Sc), induction of the phosphoprotein ScPex11p coincides with peroxisome biogenesis. We show that the ScPex11p homologue in Pichia pastoris (PpPex11p) is phosphorylated at serine 173. PpPex11p expression and phosphorylation are induced in oleate and coordinated with peroxisome biogenesis. PpPex11p transits to peroxisomes via the endoplasmic reticulum (ER). PpPex11p is unstable and ER restricted gin pex3Δ and pex19Δ cells, which are impaired in peroxisomal membrane protein biogenesis. In oleate medium, the P. pastoris mutants pex11A (constitutively unphosphorylated; S173A) and pex11D (constitutively phosphorylated; S173D) exhibit juxtaposed elongated peroxisomes (JEPs) and hyperdivided forms, respectively, although protein levels remain unchanged. In contrast with ScPex11p, the ER-to-peroxisome translocation in P. pastoris is phosphorylation independent, and the phosphorylation occurs at the peroxisome. We show that PpPex11p interacts with the peroxisome fission machinery via PpFis1p and is regulated by phosphorylation because PpPex11p and PpPex11Dp interact more strongly with PpFis1p than PpPex11Ap. Neither PpPex11p nor PpFis1p is necessary for peroxisome division in methanol medium. We propose a model for the role of PpPex11p in the regulation of peroxisome division through a phosphorylation-dependent interaction with the fission machinery, providing novel insights into peroxisome morphogenesis.     

The pas8 mutant of Pichia pastoris exhibits the peroxisomal protein import deficiencies of Zellweger syndrome cells--the PAS8 protein binds to the COOH-terminal tripeptide peroxisomal targeting signal, and is a member of the TPR protein family [published erratum appears in J Cell Biol 1993 Sep;122(5):following 1143]

We previously described the isolation of mutants of the yeast Pichia pastoris that are deficient in peroxisome assembly (pas mutants). We describe the characterization of one of these mutants, pas8, and the cloning of the PAS8 gene. The pas8 mutant is deficient for growth, but not for division or segregation of peroxisomes, or for induction of peroxisomal proteins. Two distinct peroxisomal targeting signals, PTS1 and PTS2, have been identified that are sufficient to direct proteins to the peroxisomal matrix. We show that the pas8 mutant is deficient in the import of proteins with the PTS1, but not the PTS2, targeting signal. This is the same import deficiency as that found in cells from patients with the lethal human peroxisomal disorder Zellweger syndrome. Cloning and sequencing of the PAS8 gene reveals that it is a novel member of the tetratricopeptide repeat gene family. Antibodies raised against bacterially expressed PAS8 are used to show that PAS8 is a peroxisomal, membrane-associated protein. Also, we have found that in vitro translated PAS8 protein is capable of binding the PTS1 targeting signal specifically, raising the possibility that PAS8 is a PTS1 receptor.     

Formation of the peroxisome lumen is abolished by loss of Pichia pastoris Pas7p, a zinc-binding integral membrane protein of the peroxisome.

We have cloned and sequenced PAS7, a gene required for peroxisome assembly in the yeast Pichia pastoris. The product of this gene, Pas7p, is a member of the C3HC4 superfamily of zinc-binding proteins. Point mutations that alter conserved residues of the C3HC4 motif abolish PAS7 activity and reduce zinc binding, suggesting that Pas7p binds zinc in vivo and that zinc binding is essential for PAS7 function. As with most pas mutants, pas7 cells exhibit a pronounced deficiency in import of peroxisomal matrix proteins that contain either the type 1 peroxisomal targeting signal (PTS1) or the type 2 PTS (PTS2). However, while other yeast and mammalian pas mutants accumulate ovoid, vesicular peroxisomal intermediates, loss of Pas7p leads to accumulation of membrane sheets and vesicles which lack a recognizable lumen. Thus, Pas7p appears to be essential for protein translocation into peroxisomes as well as formation of the lumen of the organelle. Consistent with these data, we find that Pas7p is an integral peroxisomal membrane protein which is entirely resistant to exogenous protease and thus appears to reside completely within the peroxisome. Our observations suggest that the function of Pas7p defines a previously unrecognized step in peroxisome assembly: formation of the peroxisome lumen. Furthermore, because the peroxisomal intermediates in the pas7 delta mutant proliferate in response to peroxisome-inducing environmental conditions, we conclude that Pas7p is not required for peroxisome proliferation.     

Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles.

We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43 degrees C but grow well at 35 degrees C. In both strains peroxisomes were completely absent in cells grown at 43 degrees C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive growth conditions (< or = 37 degrees C). However, at intermediate temperatures (38-42 degrees C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one--or infrequently a few--small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H.polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.     

Pex17p is required for import of both peroxisome membrane and lumenal proteins and interacts with Pex19p and the peroxisome targeting signal-receptor docking complex in Pichia pastoris

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Delta mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Delta mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal-receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.     

Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins.

Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.     

The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal tripeptide peroxisomal targeting signal.

The peroxisomal targeting signal 1 (PTS1), consisting of a C-terminal tripeptide (SKL and variants), directs polypeptides to the peroxisome matrix in evolutionarily diverse organisms. Previous studies in the methylotrophic yeast Pichia pastoris identified a 68 kDa protein, PAS8p, as a potential component of the PTS1 import machinery. We now report several new properties of this molecule which, taken together, show that it is the peroxisomal PTS1 receptor. (i) PAS8p is localized to and tightly associated with the cytoplasmic side of the peroxisomal membrane, (ii) peroxisomes of wild-type, but not of pas8 delta (null) mutant, P.pastoris cells bind a PTS1-containing peptide (CRYHLKPLQSKL), (iii) CRYHLKPLQSKL can be cross-linked to PAS8p after binding at the peroxisome membrane and (iv) purified PAS8p binds CRYHLKPLQSKL with high affinity (nanomolar dissociation constant). In addition, the tetratricopeptide repeat (TPR) domain of PAS8p is identified as the PTS1 binding region.     

The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1.

We report the cloning of PER6, a gene essential for peroxisome biogenesis in the methylotrophic yeast Pichia pastoris. The PER6 sequence predicts that its product Per6p is a 52-kDa polypeptide with the cysteine-rich C3HC4 motif. Per6p has significant overall sequence similarity with the human peroxisome assembly factor PAF-1, a protein that is defective in certain patients suffering from the peroxisomal disorder Zellweger syndrome, and with car1, a protein required for peroxisome biogenesis and caryogamy in the filamentous fungus Podospora anserina. In addition, the C3HC4 motif and two of the three membrane-spanning segments predicted for Per6p align with the C3HC4 motifs and the two membrane-spanning segments predicted for PAF-1 and car1. Like PAF-1, Per6p is a peroxisomal integral membrane protein. In methanol- or oleic acid-induced cells of per6 mutants, morphologically recognizable peroxisomes are absent. Instead, peroxisomal remnants are observed. In addition, peroxisomal matrix proteins are synthesized but located in the cytosol. The similarities between Per6p and PAF-1 in amino acid sequence and biochemical properties, and between mutants defective in their respective genes, suggest that Per6p is the putative yeast homolog of PAF-1.     

Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.     

Novel peroxisome clustering mutants and peroxisome biogenesis mutants of Saccharomyces cerevisiae

The goal of this research is to identify and characterize the protein machinery that functions in the intracellular translocation and assembly of peroxisomal proteins in Saccharomyces cerevisiae. Several genes encoding proteins that are essential for this process have been identified previously by Kunau and collaborators, but the mutant collection was incomplete. We have devised a positive selection procedure that identifies new mutants lacking peroxisomes or peroxisomal function. Immunofluorescence procedures for yeast were simplified so that these mutants could be rapidly and efficiently screened for those in which peroxisome biogenesis is impaired. With these tools, we have identified four complementation groups of peroxisome biogenesis mutants, and one group that appears to express reduced amounts of peroxisomal proteins. Two of our mutants lack recognizable peroxisomes, although they might contain peroxisomal membrane ghosts like those found in Zellweger syndrome. Two are selectively defective in packaging peroxisomal proteins and moreover show striking intracellular clustering of the peroxisomes. The distribution of mutants among complementation groups implies that the collection of peroxisome biogenesis mutants is still incomplete. With the procedures described, it should prove straightforward to isolate mutants from additional complementation groups.     

Rapid isolation and characterization of CHO mutants deficient in peroxisome biogenesis using the peroxisomal forms of fluorescent proteins

We isolated and characterized CHO mutants deficient in peroxisome assembly using green fluorescent protein (GFP) and blue fluorescent protein (BFP) as the fluorescent probes to study the molecular mechanism of peroxisome biogenesis. We used stable transformants of CHO cells expressing GFP appending peroxisome targeting signal-1 (PTS1) and/or peroxisome targeting signal-2 (PTS2) as the parent strains for rapid isolation of the mutants. We have obtained six peroxisome-deficient mutants by visual screening of the mislocalizations of the peroxisomal GFPs. Mutual cell fusion experiments indicated that the six mutants isolated were divided into four complementation groups. Several of the mutants obtained possessed defective genes: the PEX2 gene was defective in SK24 and PT54; the PEX5 gene in SK32 and the PEX7 gene in PT13 and PT32. BE41, which belonged to the fourth complementation group, was not determined. When peroxisomal forms of BFP were transiently expressed in mutant cells, the peroxisomal BFPs appending both PTS1 and PTS2 appeared to bypass either the PTS1 or PTS2 pathway for localization in SK32. This observation suggested that other important machinery, in addition to the PTS1 or PTS2 pathway, could be involved in peroxisome biogenesis. Thus, our approach using peroxisomal fluorescent proteins could facilitate the isolation and analysis of peroxisome-deficient CHO mutants and benefit studies on the identification and role of the genes responsible for peroxisome biogenesis.     

Chinese hamster ovary cell mutants defective in peroxisome biogenesis. Comparison to Zellweger syndrome

We have previously reported the isolation of Chinese hamster ovary (CHO) cell mutants that are defective in the biosynthesis of plasmalogens, deficient in at least two peroxisomal enzymes (dihydroxyacetonephosphate (DHAP) acyltransferase and alkyl-DHAP synthase), and in which catalase is not found within peroxisomes (Zoeller, R. A., and Raetz, C. R. H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170). We now provide further evidence that three such strains are more generally defective in peroxisome biogenesis. Electron microscopic cytochemistry revealed that the mutants did not contain recognizable peroxisomes. However, immunofluorescence microscopy using an antibody directed against peroxisomal integral membrane proteins revealed the presence of peroxisomal membrane ghosts resembling those seen in cells of patients suffering from one of the human peroxisomal disorders, Zellweger syndrome. Immunoblot analyses, using antibodies specific for peroxisomal matrix proteins, demonstrated deficiencies of peroxisomal proteins in the mutant CHO cells that were similar to those in Zellweger syndrome. Fusion of a CHO mutant with fibroblasts obtained from Zellweger patients resulted in restoration of peroxisomal dihydroxyacetonephosphate acyltransferase and peroxisomal acyl-coenzyme A oxidation activities. The hybrid cells also regained the ability to synthesize plasmenylethanolamine. Moreover, normal peroxisomes were seen by immunofluorescence in the hybrid cells. These results indicate that the hybrid cells have recovered the ability to assemble peroxisomes and that, although the mutant CHO cells are biochemically and morphologically very similar to cells from patients with Zellweger syndrome, the genetic lesions are distinct. Our somatic cell mutants should be useful in identifying factors and genes involved in peroxisome biogenesis and may aid the genetic categorization of the various peroxisomal disorders.