Proc. Natl. Acad. Sci. USAVol. 86, pp. 5419-5423, July 1989Cell Biology

Isolation of peroxisome-deficient mutants ofSaccharomyces cerevisiae

(3ioxidation/catalase/protein import machinery/Zeilweger syndrome)

RALF ERDMANN*, MARTEN VEENHUISt, DAPHNE MERTENS*, AND WOLF-H. KUNAU*t*Institute of Physiological Chemistry, Ruhr-University, 4630 Bochum 1, Federal Republic of Germany; and tLaboratory of Electron Microscopy, BiologicalCentre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands

Communicated by N. E. Tolbert, March 31, 1989 (received for review November 23, 1988)

ABSTRACT Two mutants of Saccharomyces cerevisiae af-fected in peroxisomal assembly (pas mutants) have been iso-lated and characterized. Each strain contains a single mutationthat results in (i) the inability to grow on oleic acid, (ii)accumulation of peroxisomal matrix enzymes in the cytosol,and (iu) absence of detectable peroxisomes at the ultrastruc-tural level. These lesions (pasl-l and pas2) are shown to benonallelic and recessive. Crossing of pasi-l and pas2 strainsresulted in diploid cells that had regained the ability to grow onoleic acid as sole carbon source and to form peroxisomes. Thesepas mutants may provide useful tools for future studies on themolecular mechanisms involved in peroxisomal assembly.

Eukaryotic cells maintain a proper level of biochemicalcompartmentation by means of various distinct subcellularorganelles. Peroxisomes represent a class of such organelles.They are widely distributed in eukaryotic organisms, includ-ing fungi, and are involved in a variety ofmetabolic processes(1-4). Their ubiquitous presence indicates that peroxisomesmay fulfill a number of essential functions in the cells (2-5).In recent years, the importance of peroxisomes for mamma-lian cells has also become clear (6, 7). This is especiallyemphasized in a newly recognized class of inborn humandiseases, the peroxisomal disorders (8, 9). Dysfunction ofperoxisomes in humans usually has profound clinical conse-quences.

Zellweger syndrome was the first of these peroxisomaldisorders to be reported (10) and appears to be caused by asingle recessive mutation that abolishes the import of pro-teins into the peroxisomal matrix (11). However, the com-ponents ofthe peroxisomal import machinery are still entirelyunknown.

Yeast mutations have been very successfully used for thedissection of many complex cellular phenomena, such as thecell cycle (12) and the secretory pathway (13). Since it ispossible to induce a pronounced peroxisomal proliferation inSaccharomyces cerevisiae (14), this yeast should be usefulfor the molecular analysis of the biogenesis of peroxisomes.

In an approach to analyze sorting of peroxisomal proteins,we have set out to find yeast mutants impaired in peroxisomalfunctions. Here, we report the occurrence of such mutantsamong strains of S. cerevisiae that lost the capacity to growon oleic acid. Emphasis is placed on the isolation and initialcharacterization of mutants that completely lack detectableperoxisomes.

MATERIALS AND METHODSYeast Strains. S. cerevisiae strains were used for mutant

isolation and crossing experiments: X2180-1A (MATa, mal,gal2, SUC2, CUP]); XP300-26D (a, ade2, trp5, his6, lysi,

gal2); XJB3-1B (MATa, met6, gal2); XJB3-1D (MATa, met6,gall, gal2). All strains were from W. Duntze (Bochum).

Media. Complete medium, 1% yeast extract/2% peptone/2% glucose (YPD); minimal medium, 0.67% yeast nitrogenbase without amino acids/0.5% glucose (YNB); selectivemedium, 0.67% yeast nitrogen base without amino acids/0.05% yeast extract/0.1% oleic acid plus 0.5% Tween 40(YNO) or 2% sodium acetate (YNA); presporulation medium,1% yeast extract/1% nutrient broth/5% glucose; sporulationmedium, 1% potassium acetate/0.25% yeast extract/1% glu-cose; induction medium, 0.67% yeast nitrogen base withoutamino acids/0.5% yeast extract/0.5% peptone/0.1%glu-cose/0.1% oleic acid plus 0.5% Tween 40. To supplementauxotrophic requirements, amino acids [histidine (30 ,ug/ml),lysine (30 gg/ml), methionine (30 ,ug/ml), tryptophan (20,ug/ml)] and/or a nucleotide base [adenine (20 ,ug/ml)] wereadded to minimal media. Media were solidified with 2%bacto-agar.

Mutagenesis. X2180-1A cells were grown in YNB mediumand treated with 3% ethyl methanesulfonate for 45 min at30°C (15). The survival rate was -50%. Expression ofmutations was allowed by overnight growth on YNB me-dium. After 12 hr of preincubation on selective YNO me-dium, a nystatin enrichment was performed for 90 min at anantibiotic concentration of 10 gg/ml (16). Aliquots wereplated on YNA-agar plates and subsequently replica platedon YNO-agar plates, which were incubated for 4-5 days at30°C. Strains that grew on the former type of plates but noton the latter were scored as oleic acid nonutilizers.

Genetic Analysis. Standard methods of yeast genetics (15)were used for crossing of strains, sporulation of diploids, andtetrad dissection by micromanipulation.

Analytical Procedures. Acetyl-CoA acyltransferase (3-oxoacyl-CoA thiolase; EC 2.3.1.16), catalase (EC 1.11.1.6),and fumarate hydratase (fumarase; EC 4.2.1.2) were assayedby established procedures (17). Protein was measured by themethod of Bradford (18) with bovine serum albumin asstandard.

Fractionation of Yeast Cells. Yeast cells grown on inductionmedium for 17 hr were converted to spheroplasts essentiallyas described (19). To digest the cell wall, Zymolyase 100,000(Sigma) was used at a final concentration of 25 units per g ofcells (wet weight). All subsequent steps were carried out at4°C. For homogenization, the pelleted and washed sphero-plasts were resuspended in 5 mM Mes buffer (pH 6.0)containing 0.5 mM EDTA, 0.6 M sorbitol, and 1 mM phen-ylmethylsulfonyl fluoride and lysed in a Potter-Elvehjemhomogenizer with a tightly fitting Teflon pestle by 10 strokesat 1200 rpm. After centrifugation at 1500 x g for 5 min toremove cell debris, the pellet was resuspended in the samebuffer and subjected once more to the same homogenizationprocedure. The two 1500 x g supernatants were pooled and

Abbreviation: DAB, diaminobenzidine.tTo whom reprint requests should be addressed.

5419

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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5420 Cell Biology: Erdmann et al.

centrifuged at 25,000 x g for 15 min. The resulting pellet andsupernatant were taken for enzyme measurements.

Electron Microscopy. Catalase cytochemistry was per-formed on intact cells, prefixed in 3% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.2) for 1 hr at 0C, with theconventional 3,3'-diaminobenzidine (DAB) technique de-scribed (20). Controls were performed (i) in the absence ofsubstrate, (ii) in the presence of50mM 3-amino-1,2,4-triazoleas an inhibitor of catalase activity, or (iii) in the presence of6 mM KCN to inhibit peroxidase activities.

Intact cells were fixed (or postfixed after cytochemicalstaining experiments) in 1.5% KMnO4 for 20 min at roomtemperature. After poststaining in 1% uranyl acetate indistilled water for 2-12 hr, the samples were dehydrated in agraded ethanol series and embedded in Epon 812. Ultrathinserial sections were cut with a diamond knife, mounted onFormvar-coated single hole grids, and examined in a PhilipsEM 300 without further staining. The volume fraction ofperoxisomes was determined on the sections of KMnO4-fixedcells as described (21).

RESULTSIsolation of Mutants That Cannot Utilize Oleic Acid as the

Sole Carbon Source. Mutants of S. cerevisiae were isolatedthat were unable to grow on oleic acid as the sole carbonsource. These mutants are defined in this work as strains thatin principle can grow on different nonfermentable carbonsources, such as acetate or ethanol, but not on oleic acid. Incontrast, all wild-type strains used in this study grew on oleicacid both on solid medium and liquid cultures as long as0.05% yeast extract was added to the selective medium. Thislow concentration of yeast extract did not support growtheither on agar plates or in liquid cultures without oleic acid.Additional control experiments showed that 0.5% Tween 40in the presence or absence of yeast extract also did not allowgrowth. Similar observations regarding the growth of S.cerevisiae on fatty acids were reported by Skoneczny et al.(22). Thirty-two strains that could not utilize oleic acid wererecovered among :130,000 colonies screened on replicaplates containing either acetate or oleic acid as carbonsource.

Electron Microscopical Analysis of Wild-Type and MutantCells. The subcellular morphology of the different oleic acidnonutilizing mutant strains with emphasis on the rate ofperoxisomal proliferation was investigated by electron mi-croscopy. Under inducible conditions (14, 22), peroxisomalproliferation in all mutant strains was markedly affected andranged from 1% to 10% of the peroxisomal volume fractionnormally detected in the wild-type strain under identicalconditions (14). Among the mutants, four strains were de-tected, which completely lacked recognizable peroxisomalstructures. Two examples, pasl-J and pas2 mutants (seeGenetic Analysis), are shown in Figs. 1 and 2. Fig. 1A showsthe typical substructure of wild-type S. cerevisiae grown onoleic acid as the source of carbon and energy. Besides theusual cell organelles such as nucleus, vacuoles, mitochon-dria, and endoplasmic reticulum, the cells are characterizedby the presence of many peroxisomes. These organelles aregenerally randomly distributed throughout the cytoplasm. Inaddition, many lipid droplets are present. However, perox-isomes as described above could not be detected in ultrathinsections of KMnO4-fixed pasl-l (Fig. 1C) and pas2 mutants.Serial sections of both strains were prepared, which enabledus to establish that peroxisomes or peroxisome-like organ-elles are lacking in both glucose-grown (used as an inoculum)and oleic acid-induced cells of both mutant strains. In con-trast, in the diploid crossed strain (pasl-J x pas2) peroxi-somes were again readily detectable (Fig. 1B).

As shown before (14), peroxisomes in oleic acid-growncells of S. cerevisiae are characterized by the presence ofcatalase and enzymes of the p-oxidation cycle. For thisreason, we used catalase as a marker enzyme for an addi-tional cytochemical screening for the presence of peroxi-somes. As shown in Fig. 2A, the peroxisomes in wild-typecells are densely stained after incubations for the detection ofcatalase activity with DAB and H202; similar results wereobtained in the diploid crossed strain (pasl-J x pas2) (Fig.2D). In controls, in the absence of substrate or in the presenceof aminotriazole, the microbodies remained unstained (Fig. 2A and D Insets). However, in both pas mutants catalase-positive particles could not be detected, despite extensivesearch by serial sectioning (Fig. 2 B and C). As a furthercontrol for our screening procedure, we also used wild-typecells from the exponential growth phase on glucose, whichare known to contain very few small peroxisomes. Also inthese cells, densely stained peroxisomes were readily de-tected after the DAB procedure (Fig. 2B Inset), indicatingthat our screening procedure allows the detection of evenvery small single peroxisomes. For this reason, the electronmicroscopic results indicate that peroxisomes are lacking inthe above mutant strains. As shown before (21), staining ofthe mitochondria observed in all strains tested was mostprobably due to the activity of cytochrome c peroxidase (20)because it was abolished during incubation in the presence ofKCN.

Subcellular Fractionation Studies. Biochemical analysisrevealed that the peroxisome-deficient strains still containedactivities of different enzymes of the 8-oxidation cycle. Inwild-type cells, these enzymes are located together withcatalase A in the peroxisomal matrix (14). To determine theirsubcellular localization in the mutant strains, fractionationstudies were performed. 3-Oxoacyl-CoA thiolase, a compo-nent of the peroxisomal p-oxidation system in yeast (14, 23),and catalase were used as peroxisomal marker enzymes (14),while fumarase was a measure of the intactness of mitochon-dria. These results, presented in Table 1, show that in thewild-type strain and the cross of both peroxisome-deficientmutants, the bulk of the activities of 3-oxoacyl-CoA thiolase,catalase, and fumarase were sedimentable and present in the25,000 x g pellets, indicating their particulate nature. How-ever, after differential centrifugation of lysates of both mu-tant strains the predominant part of the thiolase and catalaseactivities were detected in the 25,000 x g supernatant,whereas fumarase was still sedimentable (Table 1). Thesedata indicate that both peroxisomal enzymes, in contrast tofumarase, were present in the cytosolic compartment of theperoxisome-deficient mutants. This conclusion is in agree-ment with known properties of catalase, which is made onfree polysomes and imported into peroxisomes posttransla-tionally from the cytosol (24). Moreover, catalase is knownto be assembled in the cytosol in an active form in Zellwegercells (25-27).

Genetic Analysis. Four different strains lacking peroxi-somes were isolated by the methods described above from 32strains that did not grow on oleic acid. They were termed pasmutants because of the absence of peroxisomes and theaccumulation of peroxisomal matrix proteins in the cytosol,which implies a defect in peroxisome assembly.Complementation analysis of these four pas strains with

respect to their inability to grow on oleic acid defined threecomplementation groups. The mutants were designated pasi-1, pasl-2, pas2, and pas3. In the case of pas3, the meioticsegregation behavior revealed that it carried more than onemutation for preventing growth on oleic acid. Hence, thisstrain is not considered further in this report. In addition, thepasl-2 strain is not further characterized.

Inheritance of the mutation in pas]-J and pas2 strainsindicated that in both this trait is inherited as a single genetic

Proc. Natl. Acad. Sci. USA 86 (1989)

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I%<N,FIG. 1. Thin sections of KMnO4-fixed oleic acid grown cells of wild-type S. cerevisiae (A), the diploid cross of the two pas mutants pasl-J

and pas2 (B), and pasi-) (C) to show their overall cell morphology. Peroxisomes are evident in the wild-type and crossed strains (A and B).L, lipid droplet; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. (Bars = 0.5 Aim.)factor. The spores from 15 tetrads of pasi- and from 13tetrads of pas2 were tested. The oleic acid nonutilizer phe-

notype segregated in a 2:2 pattern except in one case. The onetetrad of pas2 showed a 3:1 segregation. Tetrads of both

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FIG. 2. Cytochemical staining of catalase activity in the cells of wild-type S. cerevisiae (A), pasl- (B), pas2 (C), and their cross (D). Afterincubation with DAB and H202, the peroxisomes are densely stained in both the wild-type (A) and the diploid crossed strain (D). (Insets) Controlsincubated in the presence of aminotriazole, which fully inhibits peroxisomal staining (arrows). Positively stained peroxisomal structures areabsent in the two mutant pas strains (B, pas]l-; C, pas2) but were readily detectable in wild-type cells in which peroxisomal synthesis is highlyrepressed by glucose (B Inset, arrow). L, lipid droplet; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. (Bars = 0.5 gm.)

Cell Biology: Erdmann et al.

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5422 Cell Biology: Erdmann et al.

Table 1. Distribution pattern of thiolase, catalase, and fumarasein the 25,000 x g supernatant and pellet fractions of cellhomogenates from wild-type and pas mutant cells of S. cerevisiae

Activity in 25,000 x g

Supernatant Pelletfraction fraction

Strain Enzyme (Al) (A2) A1/A2Wild-type Thiolase 0.% 2.82 0.3

Catalase 4.38 X 103 15.3 x 103 0.3Fumarase 4.38 15.6 0.3

pas]-] Thiolase 3.28 0.58 5.7Catalase 13.7 X 103 0.75 X 103 18.3Fumarase 5.49 20.1 0.3

pas2 Thiolase 4.15 1.05 3.9Catalase 25.1 x 103 1.6 x 103 15.7Fumarase 5.25 18.9 0.3

Diploid Thiolase 1.30 2.54 0.5strain Catalase 5.20 X 103 11.3 X 103 0.5(pasl-J Fumarase 9.02 29.1 0.3x pas2)All strains were grown in induction medium for 17 hr. This medium

contained 0.67% yeast nitrogen base without amino acids, 0.5% yeastextract, 0.5% peptone, 0.1% glucose, and 0.1% oleic acid plus 0.5%Tween 40. Cells were harvested and converted to spheroplasts, andtheir lysates were centrifuged at 1500 x g for 5 min and 25,000 x gfor 15 min. The enzymes were measured by established procedures(17) and their activities are expressed as ,umol per min per fraction.

mutants were also examined for other properties such as thecytosolic nature of peroxisomal matrix enzymes (3-oxo-acyl-CoA thiolase and catalase) and the absence of mor-phologically distinguishable peroxisomes. These traits coseg-regated with the inability to grow on oleic acid, suggesting thata single lesion was responsible. Backcrosses of the mutantstrains to wild-type cells, yielding the diploid cells from whichthe tetrads had been derived, were able to grow on oleic acid;therefore, the mutations are recessive. In addition, comple-mentation analysis showed that heterozygous diploid cells,formed by crossingpasl-] andpas2 strains, were able to growon oleic acid, whereas homozygous diploids obtained fromeach of both mutants failed to do so. The same results wereobtained with respect to the development of peroxisomes.Peroxisomes were present in the cross of pas]-l and pas2strains (Figs. 1B and 2D) but absent in homozygous strains ofboth mutants (data not shown).

DISCUSSIONThis paper describes the isolation of mutants of S. cerevisiaethat are deficient in morphologically detectable peroxisomes(pas mutants) and are unable to grow on oleic acid. Our datasuggest that pas] and pas2 define two distinct genetic locicontrolling peroxisomal assembly. These results allow alsothe designation of a general procedure for the selection ofpasmutants of this yeast. Growth on oleic acid (14) was exploitedas an initial screening procedure. Given the number ofdifferent cellular processes that influence the use ofoleic acidas carbon source, an additional biochemical screening step isclearly required. For example, in addition to pas mutants, wehave also found strains defective in individual (3-oxidationproteins but still containing peroxisomes among mutantsunable to use oleic acid. These strains, termed fox mutants(for fatty acid oxidation), will be described elsewhere. Thesecond screen utilized differential centrifugation of cell ho-mogenates to discriminate between soluble and particulateenzyme activities (Table 1), thus indicating the presence orabsence of intact peroxisomes. The cytosolic localizationof thiolase was confirmed immunocytochemically (R.E.,W.-H.K., and M.V., unpublished results). Finally, screening

results have to be confirmed by electron microscopy (Figs. 1and 2). The presence of soluble peroxisomal matrix proteinsin the pas mutants stresses the analogy of these mutants toperoxisome-deficient cells of Zellweger patients in whichdifferent peroxisomal enzymes also have been detected assoluble proteins in the cytosol (25-29). Recently, mutantswithout detectable peroxisomes have also been described ofChinese hamster ovary cells (30).A particular feature of the pas mutants described here is

that when pas]l- and pas2 strains are crossed, the resultingdiploid strain not only regains the ability to grow on oleic acidbut also forms peroxisomes again (Figs. 1B and 2D). A similarobservation is reported for fibroblasts from patients with animpairment of multiple peroxisomal functions (31, 32). Theresults in both yeast and fibroblasts seem to be inconsistentwith the known manner of peroxisome biogenesis, whichexcludes organelle formation de novo (24, 33). Accumulatedevidence suggests that peroxisomal proteins are synthesizedon free polyribosomes and are imported posttranslationallyinto preexisting organelles, which in turn grow and multiply

* by division. However, very recently, it was shown forfibroblasts of a Zellweger patient that although intact perox-isomes were not present they do contain peroxisomal ghosts(11, 34). This important finding implies that at least one defectin this disease resides in the protein import machinery.Recently, mutants defective in the assembly of other cellularstructures have been described-e.g., mas (35), mif (36),certain sec (37, 38), and rpt mutants (39).The easy accessibility of S. cerevisiae to recombinant

DNA technology makes this yeast an ideal system for agenetic approach. Therefore, the availability of pas mutantsmay greatly facilitate the molecular analysis of the variousprocesses of peroxisomal protein assembly.

We wish to thank Dr. Wolfgang Duntze (Bochum) for valuableadvice concerning several of the genetic techniques and KlasSjollema for skillful technical assistance. Part of this study wassupported by the Deutsche Forschungsgemeinschaft (Ku 329/11-2)and by the Fonds der Chemischen Industrie.

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