catalases unique cellular distribution of its four exhibits a

2006, 5(9):1490. DOI: 10.1128/EC.00113-06. Eukaryotic Cell

Marten Veenhuis and Hanspeter RottensteinerWolfgang Schliebs, Christian Würtz, Wolf-Hubert Kunau, CatalasesUnique Cellular Distribution of Its Four

Exhibits aNeurospora crassaMicrobodies: A Eukaryote without Catalase-Containing

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EUKARYOTIC CELL, Sept. 2006, p. 1490–1502 Vol. 5, No. 91535-9778/06/$08.00�0 doi:10.1128/EC.00113-06Copyright © 2006, American Society for Microbiology. All Rights Reserved.

A Eukaryote without Catalase-Containing Microbodies: Neurospora crassaExhibits a Unique Cellular Distribution of Its Four Catalases†

Wolfgang Schliebs,1‡ Christian Wurtz,1‡ Wolf-Hubert Kunau,1Marten Veenhuis,2 and Hanspeter Rottensteiner1*

Institut fur Physiologische Chemie, Abt. Systembiochemie, Ruhr-Universitat Bochum, 44780 Bochum,Germany,1 and Institute of Biology, University of Groningen, 9751 NN Haren, The Netherlands2

Received 19 April 2006/Accepted 5 July 2006

Microbodies usually house catalase to decompose hydrogen peroxide generated within the organelle by theaction of various oxidases. Here we have analyzed whether peroxisomes (i.e., catalase-containing microbodies)exist in Neurospora crassa. Three distinct catalase isoforms were identified by native catalase activity gels undervarious peroxisome-inducing conditions. Subcellular fractionation by density gradient centrifugation revealedthat most of the spectrophotometrically measured activity was present in the light upper fractions, with anadditional small peak coinciding with the peak fractions of HEX-1, the marker protein for Woronin bodies, acompartment related to the microbody family. However, neither in-gel assays nor monospecific antibodiesgenerated against the three purified catalases detected the enzymes in any dense organellar fraction. Further-more, staining of an N. crassa wild-type strain with 3,3�-diaminobenzidine and H2O2 did not lead to catalase-dependent reaction products within microbodies. Nonetheless, N. crassa does possess a gene (cat-4) whoseproduct is most similar to the peroxisomal type of monofunctional catalases. This novel protein indeedexhibited catalase activity, but was not localized to microbodies either. We conclude that N. crassa lackscatalase-containing peroxisomes, a characteristic that is probably restricted to a few filamentous fungi thatproduce little hydrogen peroxide within microbodies.

Microbodies are nearly ubiquitous organelles of the eukary-otic cell. They usually house a number of hydrogen peroxide-producing oxidases as well as catalase, which quickly removesthe hydrogen peroxide generated within microbodies (10). Pro-teins are targeted to the lumen of microbodies by either of twoperoxisomal targeting signals (PTS) (19, 48). The predominantsignal is the PTS1, a tripeptide located at the C terminus andcomposed of the amino acids SKL or conservative variantsthereof (16, 30). Depending on species, cell type, or develop-mental state, distinct types of microbodies can be prevalent,which emerge upon differential protein import. The varioustypes are termed according to their marker enzyme content,such as peroxisomes, glyoxysomes, glycosomes, or Woroninbodies (4, 24). Remarkably, filamentous ascomycetes harbor atleast two distinct types of microbodies within a single cell: (i)microbodies with a metabolic function (peroxisomes or glyoxy-somes), which house the key enzymes of the glyoxylate cycleand a complete fatty acid �-oxidation system; and (ii) theWoronin body, which is required to seal septal pores afterhyphal wounding. The Woronin body was identified as a mi-crobody-like organelle because an anti-SKL antibody specifi-cally recognized the dominant protein of this organelle (24).This protein was recently identified as HEX-1 (21, 49). HEX-1indeed harbors the PTS1 sequence SRL, aggregates within the

Woronin body, and gives rise to the typical hexagonal shape ofthis specialized organelle.

Interestingly, glyoxysomes of the filamentous fungus Neuros-pora crassa were reported to lack catalase activity. Instead,catalase activity was detected in organelles with higher densitythan glyoxysomes (25, 53). Further support for the existence ofsuch an additional microbody-like compartment was provided byWanner and Theimer (53), who subjected the N. crassa slimemutant, which lacks a rigid cell wall, to 3,3�-diaminobenzidine(DAB) staining. The DAB reaction product that is generatedupon catalase-dependent hydrogen peroxide decompositionwas absent from glyoxysomes but was found in crescent-shapedstructures in close proximity to vacuoles. However, in the reportsmentioned, the identity of this catalase-containing organelle re-mained elusive. Notably, in a more recent report, catalase activitywas detected in Woronin body-enriched fractions (49). Since insucrose density gradients the Woronin body sediments at a sig-nificantly higher density than glyoxysomes, the Woronin bodymight in fact represent the catalase-containing organelledescribed above. On the other hand, Woronin bodies arenot associated with vacuoles and their hexagonal shape doesnot resemble the prolate structures seen by Wanner andTheimer (53).

Three catalases have been described in N. crassa: catalase 1(CAT-1) and catalase 3 represent the typical large monofunc-tional catalases, whereas catalase 2 is a member of the cata-lase-peroxidase family and is possibly derived from a bacterialenzyme. All three isozymes are present throughout the N.crassa asexual life cycle, albeit to varying levels: CAT-1 ishighly abundant in conidia, CAT-2 is mainly found in aerialhyphae and conidia (37), and CAT-3 activity increases duringexponential growth and is induced under various stress condi-

* Corresponding author. Mailing address: Institut fur PhysiologischeChemie, Abt. Systembiochemie, Ruhr-Universitat Bochum, D-44780Bochum, Germany. Phone: 49-234-322-7046. Fax: 49-234-321-4266.E-mail: [email protected].

‡ W.S. and C.W. contributed equally to this study.† Supplemental material for this article may be found at http://ec

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tions (6, 33). Subcellular localization of the N. crassa catalaseshas not been thoroughly studied. Evidence exists that CAT-3 isprocessed and secreted; however, since only a little extracellu-lar CAT-3 activity has been found, it has been suggested thatmost of the enzyme is either bound to the cell wall or remainswithin the cell (34). Completion of the N. crassa genome (14)revealed a fourth putative catalase that belongs to the family ofsmall-subunit monofunctional catalases and is most similar toperoxisomal catalases of animals and yeasts (22). Thus, currentknowledge is commensurate with the existence of a peroxisomalcompartment in N. crassa that is distinct from glyoxysomes. Toclarify whether or not peroxisomes exist in N. crassa, we havethoroughly analyzed catalase activities under peroxisome-in-ducing conditions. Neither cytochemistry nor catalase activitygels supported the existence of a microbody-associated cata-lase. Likewise, the application of antibodies against the threecharacterized catalase isozymes failed to detect a lumenal cata-lase. Finally, characterization of the novel CAT-4 revealed thatthis protein is a bona fide catalase; however, this protein is nottargeted to organelles. The impact of our finding of a eu-karyote devoid of peroxisomal catalase is discussed.

MATERIALS AND METHODS

Strains and culture conditions. The Neurospora crassa wild-type strains St.Lawrence 74-OR8-1a (FGSC#988) and 74-OR23-1A (FGSC#987) were usedfor all biochemical experiments of this work. Strains Nc15 and Nc21 were gen-erated by integrating the expression constructs MF272 (green fluorescent protein[GFP] expression) (13) and CW20 (GFP-CAT-4), respectively, into the his-3locus of strain N623 (FGSC#6103) by homologous recombination, followed by ascreening of prototrophic His� transformants for expression of GFP by immu-noblotting. Strain Nc23 was similarly generated by integrating plasmid pCW22(CAT-4) into strain N623 and screening for expression of CAT-4. Wild-typeAspergillus tamarii (DSM 825; ATCC 10836) was obtained from DSMZ, Braunsch-weig, Germany. Strains were maintained on Vogel’s medium N supplementedwith 2% sucrose or, for the induction of microbodies, 1 mM oleic acid plus 1%(wt/vol) Tergitol, 40 mM acetate, or 1% (vol/vol) ethanol. All manipulationswere carried out according to standard Neurospora techniques (9).

Yeast strains used were wild-type Saccharomyces cerevisiae UTL-7A; its de-rivative, yHPR251, which harbors an integrated copy of a PTS2-DsRed construct(47); and the catalase-less strain GA1-7D cta1� ctt1� (kindly provided by C.Schuller, Max Perutz Laboratories, Vienna, Austria). Escherichia coli strainDH5� was used for all plasmid amplifications and isolations. Standard media forthe cultivation of yeast and bacterial strains were prepared as described previ-ously (43).

Plasmids and cloning procedures. To clone the reading frame of cat-4(NCU05169.2), primer pair RE1490/RE1491 was used (RE1490, 5�-AATTGGATCCATGTCTTCAAACGACGCAC-3�; RE1491 5�-AATTGAATTCTCACTCATCATCCTTCGAATC-3�). Due to our failure to amplify cat-4 from cDNAlibrary M-1 (FGSC, Kansas City, MO), genomic wild-type DNA was used astemplate in the PCR. The single 60-bp intron of the gene that follows codon 3could be gapped by primer RE1490, which contained the complementary se-quence of the first three codons, yet annealed to the sequence 3� of the intron.The PCR product was subcloned into pBluescript SK� (Stratagene), and itsidentity was verified by sequencing (MWG-BIOTECH AG, Ebersberg, Ger-many). The insert was lifted as a BamHI-EcoRI fragment and cloned intoappropriately cut pYPGE15 (5), designed for constitutive expression of CAT-4in S. cerevisiae (pCW21). To express an N-terminal GFP fusion of CAT-4 inyeast, pUG36 (42), kindly provided by J. H. Hegemann, was appropriately cut(pHPR349).

For the expression of an N-terminal GFP fusion of CAT-4 in N. crassa, GFPwas amplified from pMF272 (13) with primer pair RE1372/RE1373 (RE1372,5�-AATCTAGAATGGTGAGCAAGGGCGAG-3�; RE1373 5�-AAGGATCCCTTGTACAGCTCGTCCAT-3�), cut with XbaI and BamHI, and cloned togetherwith the BamHI-EcoRI CAT-4 fragment into XbaI-EcoRI-cut pMF272(pCW20). For the expression of untagged CAT-4 in N. crassa, the GFP openreading frame of pMF272 was replaced by the BamHI-EcoRI CAT-4 fragment(pCW22).

Purification of N. crassa catalases. All procedures were carried out at 4°C. Forthe separation and purification of three different catalases from N. crassa, allpurification steps were analyzed by native polyacrylamide gel electrophoresis(PAGE) followed by in-gel activity staining of catalases. Mechanical lysis of up to80 g of frozen mycelia grown for 48 h on acetate-containing medium was per-formed by rapid agitation with glass beads (0.1 to 0.2 mm) in 200 ml buffer A atpH 6.7 (50 mM Tris, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM benzamidine, 0.5mM dithioerythrol [DTE], 0.1 mM phenylmethylsulfonyl fluoride) using a Bead-beater (Biospec Products, Inc.). To prevent overheating of the sample during thehomogenization, the Beadbeater, with the ice water jacket installed, was oper-ated 15 times for 15 s each with intervals of 30 s. The homogenate was decantedfrom the settled glass beads, passed through four layers of gauze, and subjectedto centrifugation at 17,000 � g for 20 min. The supernatant was loaded directlyonto a coupled column system consisting of a cation-exchange column (phos-phocellulose P-11, 5 by 15.5 cm; Whatman) and a “dye-ligand” column (Blue-Sepharose Cl-6B, 1.5 by 13.5 cm). The flowthrough was subjected to anion-exchange chromatography using a Whatman DEAE cellulose DE52 column (2.5by 11 cm) equilibrated with buffer A (pH 6.7).

Bound protein including CAT-3 was eluted with a 200-ml linear gradient ofpotassium chloride (0 to 0.2 M in buffer A, pH 6.7). Peak catalase activity fractionswere pooled and loaded onto a hydroxylapatite (HA) column (2.5 by 5 cm; Bio-Rad)equilibrated in buffer B at pH 6.7 (50 mM Tris, 0.5 mM benzamidine, 0.5 mM DTE,0.1 mM PMSF). CAT-3 was eluted with a 100-ml linear gradient of 0 to 0.3 Mpotassium phosphate. Catalase-containing fractions of the flowthrough from theDE52 column equilibrated with buffer A (pH 6.7) were pooled and adjusted to a pHvalue of 7.5 by titration with a 0.1 N sodium hydroxide solution. CAT-2 and CAT-1were separated by anion-exchange chromatography using a Whatman DEAE cel-lulose DE52 column (2.5 by 10 cm) equilibrated with buffer A (pH 7.5). Only CAT-2bound to the column and was eluted with a 200-ml linear gradient of potassiumchloride (0 to 0.2 M in buffer A, pH 6.7).

CAT-1 was purified from cells grown on sucrose for 48 h by mechanicalhomogenization as described above and following the purification protocol de-scribed by Jacob and Orme-Johnson (20) with some modifications: e.g., using theBeadbeater procedure as described above for mechanical homogenization. Thefinal purification step for all three catalases, CAT-1, CAT-2, and CAT-3, was sizeexclusion chromatography performed on a Sephacryl S-300 HR column (3 by 126cm; GE Healthcare, Freiburg, Germany) equilibrated with 50 mM Tris, pH 7.5,0.5 mM benzamidine, 0.5 mM DTE, and 150 mM sodium chloride.

Generation and usage of antisera and immunoprecipitation. Antibodiesagainst N. crassa MFP (50) and TIM-23 (35), GFP (41), and yeast Cta1p (17) andPcs60p (3) were described previously. The antibody against yeast Pgk1p wascommercially obtained (Molecular Probes, Eugene, OR). Antibodies againstCAT-1, CAT-2, CAT-3, HEX-1, thiolase, and mitochondrial enoyl-coenzyme A(CoA) hydratase were generated in rabbits with solutions of native, highly pu-rified proteins. A detailed description of the purifications of the latter threeproteins will be reported elsewhere. Immunoblotting was performed according tostandard protocols. Immunoreactive complexes were detected with the ECLenhanced chemiluminescence system from GE Healthcare.

To test for the specificity of the anti-CAT-1 antibody, increasing amounts ofantiserum or preimmune serum were added to crude wild-type extract preparedfrom sucrose-grown hyphae. The soluble fractions were separated from theprecipitates by centrifugation and were subjected to an in-gel catalase assay. Forthe isolation of CAT-1 by immunoprecipitation, 30 mg of a postorganellar su-pernatant prepared from sucrose-grown mycelia was incubated with 50 �l Dyna-beads M-280 sheep anti-rabbit immunoglobulin G (Invitrogen, Karlsruhe, Ger-many) coated with anti-CAT-1 antibodies. Following triple washing withphosphate-buffered saline, beads were boiled in 50 �l of 1� sodium dodecylsulfate (SDS) sample buffer. The sample was separated by SDS-PAGE, and theprecipitated 80-kDa protein was analyzed by electrospray ionization-mass spec-trometry (ESI-MS).

Preparation of crude protein extracts, differential centrifugation, and sucrosedensity gradient centrifugation. For subcellular fractionation, cultures were in-oculated with conidia (105/ml) in Vogel�s minimal medium and were shaken (100rpm) at 30°C for 24 h before shifting hyphae to the various peroxisome-inducingconditions. Hyphae were harvested by filtration, washed with water, mixed with1 g/g wet weight quartz sand and 4 volumes of isolation buffer (150 mM Tricine,pH 7.4, 0.44 M sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EDTA), and groundwith a pestle in a mortar at 4°C. The homogenate was squeezed through fourlayers of cheesecloth and subjected to centrifugation at 2,500 � g for 5 min. Theresulting supernatant was taken as crude extract.

For differential centrifugation, 10 ml of crude extract was separated in a pelletand supernatant fraction through centrifugation at 25,000 � g for 20 min. Fordensity gradient centrifugation, 10 ml of crude extract was layered on top of a

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linear gradient of 30 to 60% (wt/wt) sucrose (in 10 mM Tricine, pH 7.4, 1 mMEDTA). The gradient was subjected to centrifugation for 90 min at 38,000 � gat 4°C in a Sorvall SV288 vertical rotor. Fractions of 1 ml were collected from thebottom to the top, and their protein concentration was determined spectropho-tometrically at 280 nm. Sucrose density was determined refractometrically.

Yeast lysates were prepared and fractionated by differential centrifugation asdescribed previously (11).

Enzyme assays. Catalase (EC 1.11.1.6), urate oxidase (EC 1.7.3.3), isocitratelyase (EC 4.1.3.1), enoyl-CoA hydratase (EC 4.2.1.17), and 3-hydroxyacyl-CoAdehydrogenase (EC 1.1.1.35) activities of MFP, fumarase (EC 4.2.1.2), andcytochrome c oxidase (1.9.3.1) were assayed by established procedures (25). Thecatalase activity of CAT-4 was similarly assayed, with the exception that variousamounts of NADPH were added to the assay mixture. In-gel catalase activityassays were carried out as described by Woodbury et al. (55). In brief, sampleswere separated on native polyacrylamide gels, which were then incubated with0.1% H2O2 for 10 min, washed twice with water, and treated with a solutioncontaining 1% FeCl3 and 1% K3(Fe(CN)6). Upon emergence of the unstainedcatalase bands, reactions were stopped by washing the gels with water.

Fluorescence microscopy. Live yeast cells were analyzed for GFP and DsRedfluorescence as described previously (41). N. crassa mycelia were similarly pre-pared. After overnight growth in Vogel’s minimal medium, hyphae were har-vested and grown overnight in induction medium (1� Vogel’s salt, 0.05% [wt/vol] Tween 40, 0.1% [wt/vol] oleic acid) at 30°C. For inspection, a suspension ofmycelia was placed on a slide, mixed with an equal volume of 1% (wt/vol)low-melting-point agarose in H2O, and sealed with a coverslip. All micrographswere recorded on a Zeiss Axioplan 2 microscope with a Zeiss Plan-Apochromat�100/1.4 oil objective and an Axiocam MR digital camera and were processedwith AxioVision 4.2 software (Zeiss, Jena, Germany).

Electron microscopy. For overall cell morphology, cells were fixed in 1.5%KMnO4 for 20 min, poststained in 0.5% uranyl acetate, subsequently dehydratedvia an ethanol series, and embedded in Epon 812. For detection of catalaseactivity, cells were prefixed in 3% glutaraldehyde in 0.1 M cacodylate buffer.Catalase activity was detected with DAB and 0.06% hydrogen peroxide as de-scribed before (52). For immunocytochemistry, the glutaraldehyde-fixed cellswere embedded in Unicryl; ultrathin sections were incubated with specific anti-CAT-1 antibodies and gold-conjugated goat anti-rabbit antiserum (54).

RESULTS

Expression of catalases under peroxisome-inducing condi-tions. To analyze whether catalase activity is induced underconditions that require microbody-borne metabolism, wild-type cells were grown for 12 h in media containing ethanol(Fig. 1, lane 1), acetate (lane 2), or oleic acid (lane 3) as thesole carbon source. Under these conditions, the glyoxylatecycle enzymes and, in the last case, the �-oxidation enzymesare induced (25). In addition, media with D-methionine (lane10), which induces the expression of D-amino acid oxidase (46),as well as media with uric acid (lane 9), which increases thelevels of urate oxidase (36), were tested for coinduction ofcatalase. Total catalase activity did not increase significantlyunder any of these conditions relative to that in control cellsthat were grown for 24 h in medium with sucrose (lane 5) asthe sole carbon source (Fig. 1A). In contrast, stationary-phasecells that had been grown for 96 h in sucrose medium (lane 8)exhibited a sevenfold increase in activity, as observed previ-ously (6, 33). Since three differentially regulated catalaseisozymes are described in N. crassa, the contribution of eachenzyme to the total catalase activity was visualized on nativeacrylamide gels that had been loaded with equal amounts ofenzyme activities. All three enzymes were clearly discernibleunder all conditions (Fig. 1B). More CAT-2 was observed inethanol- and acetate-grown cells, and as expected, CAT-3 waspredominant in cells grown to the stationary phase (33). Be-yond that we did not obtain evidence for an additional catalaseisozyme under all conditions tested.

Intracellular distribution of catalase activity. To determinethe intracellular distribution of catalase activities in an oleicacid-induced N. crassa wild-type strain, a postnuclear superna-tant devoid of cell debris and nuclei was separated on a con-tinuous sucrose density gradient (30 to 60%). The resultingfractions were assayed for the mitochondrial marker enzymesfumarase and cytochrome c oxidase as well as for the glyoxy-somal marker enzymes isocitrate lyase and the multifunctional�-oxidation enzyme (Fig. 2A). Peak Woronin body fractionswere identified immunologically by using an anti-HEX-1 anti-body (Fig. 2B). The organelles were separated well, with mi-tochondria being identified at a density of 1.19 g/cm3, glyoxy-somes at 1.21 g/cm3, and Woronin bodies at 1.26 g/cm3. Duringcell breakage, a fraction of organelles always gets ruptured,particularly the mechanically and osmotically fragile microbod-ies (25). Portions of the glyoxysomal and mitochondrial matrixenzymes were therefore also found in the lighter fractions ofthe gradient where soluble proteins and possibly organellarremnants are recovered. Most of the measurable catalase ac-tivity was found in the soluble fractions and was entirely absentfrom mitochondria and glyoxysomes. Nonetheless, a small butsignificant fraction of catalase, estimated to account for 5% oftotal activity, cosedimented with the Woronin body fractions.A similar catalase distribution was obtained when cells were

FIG. 1. N. crassa catalase activities under conditions that promoteperoxisome proliferation. N. crassa mycelia were grown in liquid me-dium containing 2% (wt/vol) sucrose for 24 h, filtered, washed, andtransferred to fresh minimal media containing the following carbonsources: 1% (vol/vol) ethanol (lane 1), 40 mM acetate (lane 2), 40 mMacetate plus 1 mM oleic acid (lane 3), 1 mM oleic acid (lane 4), and 2%sucrose (lane 5). In addition, sucrose-grown cells were supplementedwith uric acid as the sole nitrogen source (lane 9) or D-methionine asthe sole sulfur source (lane 10). After growth for 12 h, mycelia wereharvested by filtration and cell extracts were examined for total cata-lase activity (A). To test the effect of mycelial age on catalase activity,hyphae were grown in standard sucrose medium for 24 h (lane 5), 48 h(lane 6), 72 h (lane 7), or 96 h (lane 8). (B) Samples with equal catalaseactivity (40 �kat) were separated by nondenaturing PAGE and ana-lyzed for catalase activity by determining the appearance of H2O2-cleared zones after ferric cyanide/ferric chloride staining. The threecatalase isoenzymes were designated according to Michan et al. (33).

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grown on acetate or sucrose as the sole carbon source (notshown). At the same time, no isocitrate lyase or fumaraseactivity could be measured in these fractions. We also testedthe distribution of urate oxidase activity as this enzyme wasreported to cosediment with catalase in the slime mutant (53).However, urate oxidase was exclusively found in the uppersoluble fractions, indicating that urate oxidase is a solubleenzyme in N. crassa.

To analyze which of the catalase isozymes caused the ob-served catalase activity in Woronin bodies, fractions of a sim-ilar sucrose gradient were subjected to an in-gel catalase ac-tivity assay. Surprisingly, all three isozymes were only detectedin the soluble fractions, although activity was clearly measur-able also in the dense fractions (Fig. 3). Loading samples fromthe dense and soluble fractions that contained equal catalaseactivities (as measured with the liquid assay) also failed toreveal any catalase activity with the in-gel assay. These datawere interpreted to mean that the measured consumption of

FIG. 2. Subcellular localization of catalase activity by spectropho-tometry. Cell lysate of N. crassa wild-type mycelia was loaded on top ofa linear sucrose density gradient (30 to 60% [wt/wt]) and subjected tocentrifugation at 48,000 � g for 90 min. Fractions were collected from

the bottom (fraction 1) to the top (fraction 29) and were assayed formitochondrial (cytochrome c oxidase F and fumarase E), glyoxysomal(multifunctional �-oxidation enzyme ■ and isocitrate lyase �), andperoxisomal (catalase Œ and urate oxidase ‚) marker enzymes (A).Localization of Woronin bodies was determined by immunoblottingusing anti-HEX-1 antibodies (B). Dashed vertical lines indicate theorganellar peak fractions as denoted. Densities (dotted lines) andprotein concentrations (}) of each fraction are also indicated.

FIG. 3. Subcellular localization of catalase activity by an in-gel ac-tivity assay. Cell lysate of N. crassa mycelia that had been grown for12 h in oleic acid-containing medium was subjected to isopycnic 30 to60% (wt/wt) sucrose density gradient centrifugation. One-milliliterfractions were collected from the bottom (fraction 1) to the top (frac-tion 29) and measured for catalase activity by a spectrophotometricassay. The dotted line indicates the densities of each fraction (upperpanel). From the particulate fraction 5 and the supernatant fractions23, 25, and 27, aliquots with 20 nkat catalase activity each were loadedon a native 7.5% polyacrylamide gel while from the remaining frac-tions aliquots of 100 �l were loaded. After separation under nonde-naturing conditions, catalase activity staining was performed (lowerpanel).



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hydrogen peroxide in the Woronin body fractions was not dueto a catalase activity, albeit we could not entirely rule out thatthe sensitivity of the in-gel assay was too low to detect organel-lar catalase.

In an alternative approach, eventual organellar catalase ac-tivity was assayed cytochemically. Prefixed cells that had beeninduced by oleic acid were treated with H2O2 and DAB, andultrathin sections thereof were subjected to electron micros-copy. Neither glyoxysomes nor Woronin bodies contained aclear DAB reaction product that is formed in the presence ofcatalase activity (Fig. 4). Significant reaction products wereonly obtained for mitochondrial cristae, most likely caused by

an intramitochondrial peroxidase reaction (39). Notably, theDAB-positive organelles adjoined to vacuoles as described forthe N. crassa slime mutant were never observed, even whenserial ultrathin sections of a cell were inspected (see Fig. S1 inthe supplemental material). Thus, cytochemistry did not pro-vide conclusive evidence for a compartmentalized catalase.

Purification and immunological detection of the catalaseisozymes. To detect the catalase proteins in situ, we set out toproduce antibodies against the three isozymes. To that end, thethree catalase activities were purified, starting with extractsfrom acetate (CAT-2)- or sucrose (CAT-1, CAT-3)-grownwild-type cells. For CAT-1, a previously established protocolwas used (20). The various purification steps for CAT-2 andCAT-3 are summarized in Table 1. The purity of the enzymeswas analyzed by denaturing SDS-PAGE (Fig. 5A) as well as byan in-gel catalase assay (Fig. 5B). One major band was seen forcatalases 1 and 3 in the Coomassie-stained gel; the purifiedcatalase 2 sample contained one additional protein of approx-imately 46 kDa. Nonetheless, since the activity assay revealedthat each catalase gave rise to only one H2O2-reactive band,the samples were considered sufficiently pure to immunizerabbits. Tests for the specificity of the resulting antiserashowed that all antibodies were monospecific. They did notsignificantly cross-react with the other purified catalases (Fig.5C), and the antisera against CAT-2 and CAT-3 each gave riseto a single band at the expected sizes of 83 kDa and 79 kDa,respectively, in a Western blot performed with crude extractfrom oleic acid-induced wild-type mycelium (Fig. 5D). Thespecificity of the CAT-1 antiserum was further proved by animmunoprecipitation experiment. Incubation of crude wild-type extract with increasing amounts of antiserum led to de-pletion of isoform 1, but not of CAT-2 and CAT-3, in theunbound fraction (Fig. 5E).

There is some ambiguity in the literature regarding the no-menclature of the three catalase isozymes. Originally describedby Chary and Natvig (6), the catalase running most slowly inthe native polyacrylamide gel was designated as CAT-3, theslightly faster running catalase as CAT-1, and the one with thehighest mobility as CAT-2, the latter of which clearly repre-sents the catalase-peroxidase family member (NCU05770.2).

FIG. 4. In situ catalase staining. Ultrathin sections of fixed oleicacid-grown N. crassa hyphae were stained with DAB and H2O2 forcatalase activity. Visualization by electron microscopy revealed thatonly mitochondrial cristae showed an electron-dense reaction product.ER, endoplasmic reticulum; G, glyoxysomes; Lb, lipid bodies; M, mi-tochondria; N, nucleus; V, vacuole; Wb, Woronin body.

TABLE 1. Purification of three N. crassa catalases

Fraction Amt ofprotein (mg)

Total activity(mkat)

Sp act(mkat/mg) Yield (%) Fold

purified In-gel catalase activitya

Purification of CAT-3 and CAT-2Crude extract 780 1.32 1.7 100 1 CAT-1, CAT-2, CAT-3P11/Blue-Sepharose flowthrough 191 1.14 6 86.5 3.5 CAT-1, CAT-2, CAT-DE52 pH 6.7 eluate 24.3 0.35 14.4 26.5 8.5 CAT-3HA eluate 1.03 0.26 252 19.7 148 CAT-3S300 peak fraction pool 0.3 0.17 567 12.9 334 CAT-3DE52 pH 6.7 flowthrough 80.6 0.62 7.7 47 4.5 CAT-1, CAT-2 (CAT-3)DE52 pH 7.5 eluate 5 0.03 5.6 2.1 3.3 CAT-1, CAT-2S300 peak fraction pool 1 0.02 21 1.6 12.4 CAT-2

Purification of CAT-1Crude extract 2,037 0.84 0.4 100 1 CAT-1, CAT-2, CAT-3Ammonium sulfate fractionation 180 0.30 1.7 35 4.2 CAT-1 (CAT-3)DE52 eluate 6.8 0.27 39.7 32 101.2 CAT-1S300 peak fraction pool 2.0 0.18 91.7 22 229.2 CAT-1

a Parentheses indicate residual activity (presence in low amounts).



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More recently, a cat-3 knockout strain (NCU00355.2) was de-scribed by the Hansberg laboratory that surprisingly lacked thecatalase band of intermediate mobility (34), which would bereferred to as CAT-1 according to the Chary and Natvig no-menclature (6). To therefore find out at the molecular levelwhich protein is represented by the most slowly migratingcatalase, the dominant immunoprecipitated protein of approx-imately 85 kDa that remained bound to the antibody afterstringent washing was digested with trypsin and analyzed byESI-MS. Gene product NCU08791.2 (N. crassa genome re-lease 7 at the Broad Institute) could be clearly assigned to thissample (data not shown). Since this reading frame is identicalto catalase 1 (GenBank accession no. AY027545), it becameobvious that the catalase that was formerly known as isozyme3 (6) is now termed CAT-1 and vice versa. We therefore alsochose the nomenclature that was in line with the GenBankentries for CAT-1 and CAT-3.

Application of anti-CAT-1 antibodies to thin sections ofoleic acid-induced N. crassa cells revealed that subcellularcompartments were not significantly labeled. CAT-1 was ratherconcentrated at the cell wall (Fig. 6), suggesting that a portionof CAT-1 is secreted. This observation is reminiscent ofCAT-3, which is also secreted, yet only small percentage ofCAT-3 is lost to the medium (34). The antibodies were alsoused to determine the distribution of the catalases within asucrose density gradient. CAT-2 and CAT-3 did not enter the

FIG. 5. Immunologic analysis of three purified catalase isoenzymes from N. crassa. (A and B) Determination of purity. For each isoenzyme,the gel filtration fraction with the highest specific catalase activity was analyzed by denaturing (A) and nondenaturing (B) PAGE. Catalases werevisualized by Coomassie (A) and in-gel activity (B) staining, respectively. The sizes as predicted by the genome sequence are as follows: CAT-1,85.5 kDa; CAT-2, 83.4 kDa; CAT-3, 79.2 kDa. (C to E) Specificity of antisera directed against the individual purified catalases. (C) Equal amounts(5 �g) of purified catalase isoenzymes were subjected to Western blot analysis. Primary rabbit antisera were used at dilutions of 1:1,000 and incombination with alkaline phosphatase-conjugated goat anti-rabbit antibodies (�-CAT-1, �-CAT-2, and �-CAT-3). (D) Crude extract (10 �g) fromoleic acid-induced wild-type mycelium was analyzed by Western blotting using anti-CAT-2 and anti-CAT-3 antibodies (dilution of 1:20,000).(E) Immunoprecipitation of CAT-1. Increasing amounts of CAT-1 antiserum or preimmune serum were added to crude extract (200 �kat catalaseactivity), incubated for 1 h, and subjected to centrifugation. The soluble fraction was analyzed for the presence of catalase isoenzymes by an in-gelactivity assay.

FIG. 6. Immunocytochemical localization of CAT-1. Ultrathin sec-tions of glutaraldehyde-fixed mycelia of N. crassa that had been grownon sucrose-containing medium were decorated with anti-CAT-1 anti-bodies and gold-conjugated goat anti-rabbit antiserum. G, glyoxy-somes; Lb, lipid bodies; M, mitochondria; V, vacuole; Wb, Woroninbodies.



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gradient and were localized to the soluble fractions (Fig. 7A).The same was true for CAT-1 (Fig. 7B). Taken together, noneof the three measurable catalase activities was present withinmicrobodies.

Characterization of a novel N. crassa catalase. We and oth-ers (22) have searched the N. crassa genome sequence for thepresence of catalase isozymes. Interestingly, there is indeed afourth open reading frame with strong similarity to that for thecatalases, NCU05169.2, hereafter referred to as cat-4 (Fig. 8A).Since under various growth conditions and developmental statesonly three distinct activities were detectable in native gels (Fig. 1),there was no evidence for a fourth catalase being produced in N.crassa. To determine experimentally whether this reading frameencodes a true catalase, we set out to amplify cat-4 from a cDNAlibrary (FGSC, Kansas City, MO). However, this approach failed,probably due to a (very) low expression rate of this reading frame.We therefore amplified CAT-4 from genomic DNA by using aprimer pair that allowed synthesis of the intron-less gene (seeMaterials and Methods for details). cat-4 was then cloned into anexpression vector designed to heterologously express N. crassaCAT-4 in S. cerevisiae. Since the endogenous yeast catalases arecompletely repressed in the presence of 2% glucose (8, 40), cata-lase activity eventually measured under such conditions in thetransformants must stem from the expressed cat-4 gene.

A high catalase activity (461 � 41 U/mg protein) was indeedmeasured in the wild-type S. cerevisiae strain expressingCAT-4, whereas the wild-type strain transformed with theempty vector control exhibited only very low catalase activity(8 � 0.7 U/mg protein). Endogenous catalase activity (218 �11 U/mg protein) was measured in the latter strain upon in-duction by oleic acid (Fig. 8B). Expression of the cat-4 gene ina yeast strain devoid of Cta1p and Ctt1p, its endogenous twocatalases, gave rise to similar catalase activities (578 � 85U/mg protein), thereby demonstrating that CAT-4 is a bonafide catalase (Fig. 8B). The observed rapid decrease in CAT-4activity was prevented by the addition of NADPH (Fig. 8C),indicating that NADPH effectively protected CAT-4 againstinactivation by its substrate, H2O2, as has been reported forother catalases (26). To see whether our highly specific anti-body against S. cerevisiae peroxisomal catalase A (Cta1p) rec-ognizes CAT-4, the same protein extracts were analyzed byimmunoblotting. A band of the expected size for CAT-4 wasclearly visible in strains harboring the cat-4 expression con-struct, but not in the empty vector control strains (Fig. 8D). Atthe same time, antibodies against the N. crassa catalasesCAT-1, CAT-2, and CAT-3 failed to detect CAT-4 (notshown). Crude protein extracts from N. crassa wild-type my-celia grown on Vogel’s sucrose medium or oleic acid-contain-ing medium were also analyzed for the presence of CAT-4. Theyeast Cta1p antibody did not recognize any protein in thesesamples (Fig. 8D) or in the gradient fractions (Fig. 7B), indi-cating that CAT-4 is not expressed or is only very weaklyexpressed in N. crassa under the tested conditions.

A phylogenetic analysis of catalases conducted by Johnson etal. (22) revealed that CAT-4 belongs to the family of smallmonofunctional catalases that are found in bacteria, animals,and fungi. Since this clade includes all known peroxisomalcatalases, it was tempting to assume that CAT-4 is also aperoxisomal protein. Most peroxisomal catalases harbor a C-terminal peroxisomal targeting signal (PTS1) (15, 28, 38).However, the three C-terminal amino acid residues ofCAT-4 are all acidic (DDE) and drop out of representing aPTS1 (SKL or conserved variants thereof). A PTS2 se-quence (H/R-L-X5-H-L) within the N-terminal 50 amino

FIG. 7. Subcellular localization of catalases by immunoblot analysisof sucrose density gradient fractions. Cell lysates of oleic acid-inducedwild-type mycelia were layered on top of a 30 to 60% (wt/wt) sucrosegradient and subjected to isopycnic centrifugation. Fractions were col-lected from the bottom (fraction 1) to the top (fraction 28) of thegradient, and equal amounts of protein from the indicated fractionswere analyzed by SDS-PAGE and immunoblotting. The followingmarker enzymes were analyzed: enoyl-CoA hydratase (mito ECH) formitochondria; multifunctional �-oxidation enzyme (MFP) and 3-keto-acyl-CoA thiolase (thiolase) for glyoxysomes; and HEX-1 for Woroninbodies, identified as the dominant 21-kDa protein in the Coomassie-stained gel (arrow). Antibodies directed against CAT-2 and CAT-3revealed that both catalases are localized to the cytosol (A). A similargradient with 33 fractions was used to determine the localization ofCAT-1 (B), except that HEX-1 was detected immunologically andTIM-23 served as a mitochondrial marker protein. The additionalappearance of HEX-1 in the soluble fractions is due to the fraction ofWoronin bodies that were ruptured during cell breakage. CAT-4 wasnot detected in any of the fractions with the cross-reacting anti-yeastCta1p antibody.



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FIG. 8. CAT-4 is a novel catalase from N. crassa. (A) Sequence alignment of NcCAT-4 (NCU05169.2) with S. cerevisiae Cta1p (ScCta1p). The amino acidsequences were aligned using CLUSTALW with its default parameters (http://www.ebi.ac.uk/clustalw/). Asterisks denote identical residues, double dots (:) andsingle dots denote positions with conserved and semiconserved substitutions, respectively (7). The consensus signatures for protoheme binding and the proximalactive site (PROSITE; http://ca.expasy.org/prosite/) as well as the seven-element fingerprint of the catalase protein family (http://umber.sbs.man.ac.uk/dbbrowser/sprint/) are highlighted. The targeting signal of Cta1p according to Kragler et al. (28) is also denoted (PTS1). (B) Catalase enzyme activity assays. S. cerevisiaewild-type strain UTL7-A and the catalase-less mutant strain GA1-7D ctt1� cta1� were transformed with a plasmid designed to heterologously express CAT-4from the constitutive PGK1 promoter (CAT-4) or as control with the empty vector (vector). Strains were grown in the presence of 2% glucose or 0.1% oleic acidas indicated in the legend and assayed for catalase activity. Endogenous yeast catalase activity was only measurable in oleic acid-induced cells (}), whereas CAT-4was active in glucose-grown wild-type (Œ) and cta1� ctt1� mutant (E) cells. (C) Protection of CAT-4 by NADPH. Catalase activity was determined as describedfor panel B, but increasing amounts of NADPH were added to the assay mixture. (D) Immunological detection and expression of CAT-4. The same yeast strainsas well as N. crassa wild-type mycelia grown on sucrose or oleic acid were processed for SDS-PAGE and Western blot analysis. Anti-Cta1p antibodies were usedat a dilution of 1:10,000 in combination with the ECL detection system.

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acids is also missing. Notably, though, S. cerevisiae catalaseCta1p is imported into peroxisomes in the absence of itsC-terminal targeting signal, since it additionally possessesan internal targeting signal (28). Thus, despite lacking aneye-catching PTS, it remained entirely possible that CAT-4is targeted to microbodies.

We therefore determined the subcellular localization of aGFP-CAT-4 fusion protein in an S. cerevisiae strain that ex-pressed PTS2-DsRed, a synthetic peroxisomal marker protein(47). As expected, synthesis of GFP-SKL gave rise to a punc-tate staining pattern that is congruent with that of PTS2-DsRed, showing that the appended PTS1 directed GFP toperoxisomes. In contrast, diffuse fluorescence was observedwhen GFP-CAT-4 or GFP alone was expressed (Fig. 9A). Thediffuse staining obtained with the latter protein could not havebeen caused by a degradation product of GFP-CAT-4, sinceWestern blotting demonstrated the appropriate expression ofthe full-length fusion protein (Fig. 9B). Notably, though, theGFP-CAT-4 fusion protein was enzymatically inactive. Totherefore exclude that the GFP tag compromised the localiza-tion of CAT-4, the untagged enzyme was expressed in the ctt1�cta1� mutant and its distribution between a 25,000 � g or-ganellar pellet and the corresponding supernatant was deter-mined. All catalase activity was recovered from the superna-tant, whereas the peroxisomal matrix enzyme Pcs60p wasdetected in the pellet fraction (Fig. 9D). As control, yeastCta1p was recovered from the organellar pellet fraction of awild-type strain (Fig. 9C). Thus, CAT-4 did not possess anyperoxisomal targeting information; rather it was located in thecytosol.

The presented localization data strongly support the no-tion that CAT-4 is a cytosolic enzyme; nonetheless, a remotepossibility was that N. crassa maintains a distinct and uniqueperoxisomal import mechanism for CAT-4. A similar GFP-CAT-4 fusion protein was therefore also expressed in N.crassa (Fig. 10B). Not only GFP but also GFP-CAT-4 wasuniformly distributed throughout hyphae, and Woroninbodies did not contain GFP-CAT-4 (Fig. 10A). Finally, awild-type strain overexpressing untagged CAT-4 was gener-ated. Its total catalase activity (70 U/mg protein), measured

FIG. 9. CAT-4 is localized to the cytosol when expressed in S.cerevisiae. (A) Fluorescence microscopy of GFP-CAT-4-expressing S.cerevisiae cells. Yeast strain yHPR251 expressing the synthetic peroxi-somal marker protein PTS2-DsRed was transformed with plasmidsdesigned to express GFP-CAT-4, GFP-SKL or GFP. Strains weregrown on solid oleic acid-containing medium for 2 days and weresubsequently examined for GFP and DsRed fluorescence. Colocaliza-tion with PTS2-DsRed indicates peroxisomal targeting of the GFPfusion proteins. Nomarski images demonstrate the structural integrityof the cells. (B) Expression of GFP-CAT-4. Correct expression of GFPand the full-length CAT-4 fusion protein in yeast was determined byWestern blotting using anti-GFP antibodies. (C and D) Differentialcentrifugation. Postnuclear supernatants (PNS) of an oleic acid-in-duced wild-type strain (C) and the cta1� ctt1� mutant expressingCAT-4 (D) were separated into a 25,000 � g organellar pellet (OP)and a supernatant (S) fraction. Fractions were assayed (upper panels)for catalase activity and (lower panels) for the presence of cytosolicPgk1p, peroxisomal Pcs60p, and Cta1p (C) or CAT-4 (D) by Westernblot analysis. Activities measured in the PNS fractions were taken as100%.



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in a postnuclear supernatant, exceeded that of the wild-typestrain (18 U/mg protein) by a factor of 4, demonstrating thatexpression of CAT-4 in N. crassa gave rise to an activeenzyme. Subcellular fractionation of these extracts showedthat CAT-4 was exclusively present in the supernatant, whilea significant portion of the glyoxysomal marker protein MFPwas recovered from the organellar pellet fraction (Fig.10D). A similar distribution of MFP was obtained in thewild-type control strain (Fig. 10C). The combined resultstherefore demonstrated that CAT-4 is localized to the cy-tosol and not imported into microbodies.

Appearance of peroxisomal catalase in other filamentousfungi. To analyze whether microbodies of other Euascomyce-tes also lack catalase activity, potential catalases from fungiwith an available genome sequence were scrutinized for thepresence of peroxisomal targeting signals. Close relatives of N.crassa from the Sordariomycete family including Fusarium gra-minearum and Podospora anserina do possess potential cata-lases of the peroxisomal family (i.e., small monofunctionalcatalases) with high similarity to CAT-4, and these proteinsalso lack obvious C-terminal PTS sequences (see Fig. S2 in thesupplemental material). In contrast, one of the four catalasesof the Eurotiomycete Aspergillus is a small-subunit catalase(23) with potential PTS1 sequences in all sequenced Aspergillusspecies (ARL in A. nidulans, A. oryzae, and A. terreus and SRLin A. fumigatus). We therefore tested whether Aspergillus in-deed contains peroxisomal catalase. To that end, cell lysate ofoleic acid-induced A. tamarii mycelia was separated by sucrosedensity gradient centrifugation and the resulting fractions wereassayed for catalase activity by a spectrophotometric assay.When compared with the distribution of marker enzymes, itbecame evident that catalase cosedimented with the multifunc-tional protein (Fig. 11, upper panel). An in-gel activity assayshowed that one isoform of catalase was indeed present in thepeak microbody fractions (Fig. 11, lower panel). Furthermore,the genome sequences of several other Eurotiomycetes, suchas Histoplasma capsulatum, Coccidioides immitis, Sclerotiniasclerotiorum, and Botrytis cinerea also revealed the presence ofputative peroxisomal catalases with a PTS1 (see Fig. S2 in thesupplemental material). Thus, the absence of catalase-contain-ing microbodies is probably restricted to a few filamentousfungi of the Sordariomycetes.

DISCUSSION

The filamentous Euascomycete N. crassa is thought to possessthree types of microbodies within a single cell: glyoxysomes,

FIG. 10. CAT-4 is a cytosolic enzyme in N. crassa. (A) Localizationof GFP-CAT-4 upon expression in N. crassa mycelia. Plasmids de-signed to express GFP-CAT-4 or GFP were integrated into the his3locus of N. crassa strain N623. Prior to inspection, transformants weregrown in minimal medium containing sucrose as the sole carbonsource. The left segment of the hypha was damaged during prepara-tion, and the cytoplasm leaked out. A Woronin body sealing the septal

pore is discernible. Wb, Woronin body; S, septum. (B) Expression ofGFP-CAT-4. Correct expression of GFP and the full-length CAT-4fusion protein in N. crassa was determined by Western blotting usinganti-GFP antibodies. (C and D) Differential centrifugation. Post-nuclear supernatants (PNS) of an oleic acid-induced wild-type strain(C) and a wild-type strain ectopically expressing CAT-4 (D) wereseparated into a 25,000 � g organellar pellet (OP) and a supernatant(S) fraction. Fractions were assayed (upper panels) for catalase activityand (lower panels) for the presence of peroxisomal MFP and CAT-4by Western blot analysis. Activities measured in the PNS fractionswere taken as 100%.



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Woronin bodies, and a distinct catalase-containing compart-ment. Here we have shown that N. crassa lacks particulatecatalase. As a consequence, the existence of a peroxisomalcompartment in N. crassa can be disregarded. The observationmade was rather surprising in light of the fact that catalase wasactually the first established marker enzyme for microbodies(10). Indeed, all catalase-containing eukaryotes examined sofar also contained a peroxisomal isoform of this enzyme.

The catalase activities that were detected in oleic acid-in-duced N. crassa mycelia were identical to the three describedcatalase isoforms, CAT-1, CAT-2, and CAT-3 (33, 37). Toaddress their subcellular localization, all three catalases werepurified and used for the preparation of monospecific antibod-ies. These immunoglobulins did not detect any of the threecatalases in the organellar fractions of sucrose density gradi-ents; instead, they were localized to the cytosol. Electron mi-croscopy revealed that CAT-1 additionally associated with thecell wall, as was also reported for CAT-3 (34). Thus, it appearsadvantageous for filamentous fungi to furnish the extracellularmedium with catalase so as to detoxify peroxide already out-side the mycelia.

Interestingly, also N. crassa contains one gene (cat-4) thatdoes encode a peroxisomal type of catalase. The deducedamino acid sequence of cat-4 features consensus signatures for

both protoheme binding as well as the catalase active site. Weshowed that CAT-4 indeed represents a bona fide catalase, asit was able to decompose hydrogen peroxide in a heterologousenvironment. Notably, the enzyme quickly lost its activity uponaddition of its substrate, hydrogen peroxide, an effect that wasnot observed for yeast catalase. Several catalases are suscepti-ble to inactivation by their own substrate, H2O2, but this canbe largely prevented by bound NADPH (26). Addition ofNADPH indeed stimulated the activity of CAT-4, probably bypreventing it from oxidative damage. The CAT-4 sequence didnot reveal any obvious peroxisomal targeting signals, suggest-ing that this catalase is not targeted to peroxisomes. However,a few peroxisomal matrix proteins such as yeast acyl-CoA ox-idase (27, 44) lack either of the two prevalent targeting signals,PTS1 and PTS2. Also N. crassa glyoxysomes contain at leastone such protein, the multifunctional enzyme (MFP) encodedby the fox-2 gene (12). It is worth noting that the orthologouscatalase from S. cerevisiae can use an ill-defined internal tar-geting signal in lieu of its C-terminal PTS1 (28). These alter-native avenues of import notwithstanding, ectopic expressionof CAT-4 in N. crassa did not result in targeting to any intra-cellular membrane-bound structure. Furthermore, CAT-4 wasalso not localized to peroxisomes upon heterologous expres-sion in S. cerevisiae.

Although it proved possible to demonstrate catalase activityfor CAT-4 in both N. crassa and S. cerevisiae, we did not obtainevidence for endogenous CAT-4 being expressed in N. crassa:anti-yeast Cta1p antibodies, which did recognize CAT-4 whenexpressed heterologously in yeast or ectopically in N. crassa,failed to yield a signal when applied to protein extracts fromwild-type mycelia grown in sucrose or oleic acid-containingmedium. Furthermore, under various peroxisome-inducingconditions only CAT-1, CAT-2, and CAT-3 were discernible innative gels stained for catalase activity. Obviously, our studiesstill leave room for environmental conditions under whichCAT-4 will be expressed. However, important for our corestatement is the fact that ectopic expression of CAT-4 wasfeasible and this led to a cytosolic localization of the protein.

The previously reported microbody-like organelle of highdensity with apparent catalase activity (25, 53) is likely tocoincide with the Woronin body, since spectrophotometric as-says at 240 nm conducted by us and others (49) indeed mea-sured a weak activity associated with Woronin bodies. How-ever, several findings strongly argue against the presence ofcatalase in this organelle: DAB treatment of N. crassa hyphaedid not reveal conclusive evidence for the presence of a cata-lase in Woronin bodies (or any other organelle), albeit the lowsensitivity of this method would not detect very small amountsof catalase. Also native gel assays could not confirm this activ-ity. Specific immunologic detection of the three catalase iso-forms CAT-1, CAT-2, and CAT-3 showed that these proteinsare not associated with any intracellular compartment. Consis-tent with this result, the coding sequences of CAT-1, -2, and -3all lack PTS1 or PTS2 sequences. Finally, a GFP fusion of thenovel CAT-4 was also shown to be localized to the cytosol. Oneplausible explanation for the spectrophotometric hydrogenperoxide breakdown could be the presence of an unidentifiedperoxidase associated with the Woronin body fraction. Little isknown about the protein composition of this organelle. Itsdominant protein, HEX-1, possessed neither catalase nor per-

FIG. 11. Subcellular localization of catalases from Aspergillus tamariiby an in-gel activity assay. Cell lysate of oleic acid-induced wild-type A.tamarii mycelia was separated on a 30 to 60% (wt/wt) sucrose densitygradient. Fractions were collected from the bottom (fraction 1) to thetop (fraction 30) of the gradient and (upper panel) examined for thedistribution of the following enzyme activities: fumarase (E), multi-functional �-oxidation enzyme (Œ), and catalase (}). Densities (dottedline) of each fraction are also indicated. The lower panel shows anin-gel catalase activity assay in which equal amounts of protein (10 �g)from the indicated fractions had been separated by native PAGE.Catalase is clearly discernible in the peroxisomal fractions.



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oxidase activity when enriched by cation-exchange/gel filtrationchromatography (data not shown).

Why is catalase dispensable for Neurospora microbodies?This is likely to be a direct consequence of the organellarenzyme content of this organism. The glyoxysomal �-oxidationsystem of N. crassa is unusual in that the first step is catalyzedby an acyl-CoA dehydrogenase instead of an acyl-CoA oxidase(25). As a consequence, hydrogen peroxide is not formedwithin glyoxysomes during that process. Furthermore, theH2O2-generating urate oxidase activity was localized to thecytosol. In all mammals that do express urate oxidase, thisenzyme involved in purine metabolism is localized to peroxi-somes (18). Even urate oxidases from Aspergillus are peroxi-somal and bear PTS1 sequences, while the probable Neuros-pora ortholog, NCU07853.2, does not (for alignments of fungalurate oxidases, see Fig. S3 in the supplemental material). Itwill be interesting to see whether H2O2-producing oxidases areat all present in glyoxysomes of N. crassa. One candidate isD-amino acid oxidase, since its putative reading frame,NCU06558.2, harbors the canonical PTS1 tripeptide SKL at itsC terminus. In any case, it is conceivable that due to theabsence of the main sources of H2O2 production, particularlyfatty acid �-oxidation, moderate amounts of H2O2 eventuallyproduced inside glyoxysomes are tolerated even without a cata-lase.

In line with this perception was the observation that Aspergil-lus strains do contain peroxisomal catalase, since this genusexploits the typical acyl-CoA oxidase for the first step in per-oxisomal �-oxidation (32, 51). This difference in catalase lo-calization is remarkable in so far as Neurospora and Aspergillusare both members of the subphylum Pezizomycotina. A closerlook at the phylogenetic tree reveals that Neurospora belongsto the class of Sordariomycetes, whereas the Aspergillus genusbelongs to the Eurotiomycetes. A comparison of the putativeorthologs of CAT-4 from Sordariomycetes with a sequencedgenome revealed that none of these catalases possess a PTS1signal, whereas those of the Eurotiomycetes class did. Wetherefore speculate that the Sordariomycetes lineage lost itsperoxisomal catalase in the course of evolution because itbecame redundant as a consequence of reduced hydrogen per-oxide production within microbodies. In light of the excep-tional role of this organelle, it should be noted that the micro-body-related glycosomes of trypanosomes harboring most ofthe glycolysis enzymes are also free of catalase. However, theseparasites do not possess any catalase and use trypanothione-dependent peroxidases as an alternative system for peroxideremoval (2, 45). Also the insect cell lines Sf9 and Sf21, derivedfrom Spodoptera frugiperda pupal ovarian tissue, were reportedto lack catalase (1, 29). Nonetheless, since insects do possessperoxisomal catalase at particular stages of development (31),the observations made merely reflect conditions under whichperoxisomal catalase was not expressed. In the case of N.crassa, however, the catalases are intrinsically absent from mi-crobodies and, as a consequence, peroxisomes are truly missingin this and possibly a few other closely related organisms.

ACKNOWLEDGMENTS

We want to thank C. Schuller for the catalase-less yeast strain, M.Freitag for plasmid MF272, H. F. Tabak for the Cta1p antibody, and

D. Mokranjac for the TIM-23 antibody. Special thanks go to B.Warscheid for the ESI-MS analysis.

This work was supported by Deutsche Forschungsgemeinschaftgrant SFB480 (to H.R. and W.-H.K.).

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catalases unique cellular distribution of its four exhibits a

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