Expression and subcellular localization of Candida tropicalis catalase in catalase gene disruptants of Saccharomyces cerevisiae

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 85, No. 6, 571-578.

1998

Expression and Subcellular Localization of Candida tropicalis Catalase in Catalase Gene Disruptants of Saccharomyces cerevisiae HJROSHI

KINOSHITA,’ MITSUYOSHI UEDA,’ HARUYUKI ATOMI,’ NORIKO HASHIMOTO,* KEIKO KOBAYASHI,2 TOMOKO YOSHIDA,2 NAOMI KAMASAWA,2 MASAKO 0SUMI,2 AND ATSUO TANAKA**

Laboratory of Applied Biological Chemistry, Department of Synthetic Chemtitry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606~8501* and Department of Chemical and Biological Sciences, FacuIty of Science, Japan Women 3 University, 2-8-I Mejirodai, Bunkyo-ku, Tokyo 112-8681,2 Japan Received 15 December 1997/Accepted 20 February 1998

We previously reported the construction of an overexpression system of Candida tropicalis peroxisomal catslase (CTC) gene in the Saccharomyces cerevisiae cytoplasm using the GAL 7 promoter (Kinoshita et al., Appl. Microbial. Biotechnol., 40: 682-686, 1994). To study the mechanism of the subcellular localization of peroxisomal catalase which is composed of four identical subunits and four heme molecules, the system was improved by changing the promoter used in order to express the catalase subunit accompanying the proliferation of peroxisomes in S. cerevisiae cells (S. cerevisiae MAT strain), in which catalase A and catalase T genes were disrupted. On native-PAGE, besides the functional tetrameric form (220 kDa), other larger multimeric forms of recombinant CTC were observed in the cell-free extract. It was clearly demonstrated that only the tetrameric form, among the various forms, contained heme and exhibited catalase activity. Subcellular fractionation and protease protection experiments indicated that only the functionally active tetrameric form was present inside the peroxisomes even under conditions of overexpression of the catalase subunit, in which different multimeric forms including the active tetrameric form were produced outside the peroxisomes. The results indicated that C. tropicalis catalase subunit and heme stoichiometrically localize even in S. cerevisiae peroxisomes, suggesting a common regulatory role of peroxisomes themselves for the transport of the oligomerit form of catalase beyond yeast species. [Key words: yeast, peroxisomes,

catalase, heme, transport]

Peroxisomes, which are one of the subcellular organelles of eukaryotic cells, contain metabolic systems including the fatty acid j-oxidation system (l-4). This organelle in Saccharomyces cerevisiae proliferates when the yeast is growing in the presence of carbon sources such as oleic acid, accompanying the induction of many peroxisomal enzymes. Catalase is a representative marker enzyme of peroxisomes and degrades hydrogen peroxide, which is produced at the first step of fatty acid ,%oxidation. The genetic information of catalase is located on a chromosome in the nucleus (5, 6), and most of the catalase synthesized on free polysomes in the cytosol is transported posttranslationally into pre-existing peroxisomes (3, 7, 8). The signals for peroxisomal localization of catalase A in S. cerevisiae (9, 10) and of catalase in Hansenulapolymorpha (11) have been identified, and PAS10 has been recognized as one of the components essential for the peroxisomal localization of catalase (12). However, the mechanism of its transport has not been clarified completely. It has been considered that peroxisomal catalase assembles with heme inside peroxisomes (13). However, in addition to the fact that catalase exists in the cytosol of S. cerevisiae (catalase T), it was found that catalase could assemble into a tetrameric form in the cytosol of guinea pig hepatocytes and liver, peroxisome-deficient (Zellweger syndrome) human cells (14-17), and pasI0

mutants of S. cerevisiae (12). These results indicate the ability of catalase to assemble in the cytosol, and that peroxisomes are not necessary for its assembly into the active tetrameric form. To study the general or actual function of peroxisomes on catalase assembly and transport beyond species, we first established an expression system for Candida tropicalis catalase (CTC) gene using the galactose-inducible GAL7 promoter in S. cerevisiae. It was revealed that purified recombinant CTC (rCTC) was identical to native CTC, and that rCTC could bind with heme and assemble into a tetrameric form in the cytosol in the absence of peroxisome proliferation (18). This paper deals with the construction of an overexpression system of catalase subunits under conditions of peroxisome proliferation, and investigation of the subcellular localization of the oligomeric form of catalase in peroxisomes, and of the functions of peroxisomes themselves on the assembly and transport of catalase. The results obtained suggest the possibility that catalase subunits and heme may be stoichiometrically (at a ratio of 1 : 1, including the active tetrameric form) transported across the peroxisomal membrane, and that peroxisomes may not allow the passage of excessive amounts of catalase subunits, or its multimeric forms, except for the tetrameric form. MATERIALS

AND METHODS

Strains Escherichia coli strains DH 5a [F- , endAl, hsdR17 (rk-. mkm), supE44, thi-1, P, recA1, gyrA96, AlacU169(#80 lacZAM15)] and HBlOl [F-, leuB6, thi-1 lacY1, hsdS20 (rkp, mk- ) recA13 rpsL20, ara-14, gaiK2,

* Corresponding author. Enzyme. Catalase (EC 1.11.1.6); isocitrate lyase (EC 4.1.3.1) Abbreviations: CTC, Candida tropicalis catalase; rCTC, recombinant C. tropic& catalase; ICL, isocitrate lyase; G7, GAL7 571

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~~1-5, mtl-1, supE44 mcrA+, mcrB-] were used for amplification of the plasmids constructed. For expression of CTC, S. cerevisiae strain MT81 (MATa ade his3 leu2 trpl ura3) was transformed with plasmid pMT34( - G7)ICL-CTC, which contained the CTC genomic DNA and the ICL (isocitrate lyase) promoter region of Candida tropicalis (19). Catalase A gene (20) and catalase T gene (21) disruptants of S. cerevisiae (AAAT strain) were prepared by homologous recombination, using LEU2 and HIS3 as the selective markers. Growth media E. coli was grown in LB medium (1% tryptone, 0.5% yeast extract, 0.5% sodium chloride; w/v) containing 0.1% glucose. YPD medium (1% yeast extract, 1% peptone, and 2% glucose) was used for the precultivation of yeast. To induce CTC gene expression, S. cerevisiae was grown in YPO medium composed of 1% yeast extract, 1% peptone, and 0.5% oleic acid. This medium was also used for the preparation of the yeast cells used for subcellular fractination. Galactose medium was described in the previous paper (18). Construction of the expression plasmid pMT34( - G7)ICL-CTC The subcloned catalase gene of C. tropicalis in pUC19 was used as a template for polymerase chain reaction (PCR). The primer used to introduce an Ncol site, which hybridized with the 5’-terminal region, was 5’-GACCATGGCTCCTACCTTCAAAA-3’. The primer for a Hind111 site, hybridizing with the 3’-terminal region, was 5’-GCAAGCTTTAGGTATCGTGGTGCTA TGA-3’. Amplified fragments were ligated with the promoter region (1570 bp) of the ICL Bl clone (19, 22) and were subcloned into the BamHI-Hind111 gap of pUC19. This plasmid was digested with BamHI and Seal, and the prepared fragments were cloned into the BamHIPvuII gap of the shuttle vector plasmid pMT34-(+3) (pMT34( - G7)-ICL-CTC). As a result, GAL7 promoter in the original plasmid pMT34-(+3) was excluded (23). The plasmid pMT34( - G7)-ICL-CTC was first amplified in E. coli and used to transform S. cerevisiae using the lithium acetate method (24). Subcellular fractionation A method established previously (25) was used for the subcellular fractionation. After homogenization of protoplasts, the peroxisome-containing fraction was obtained by centrifugation at 20,000 x g. Protease and detergent treatments A reaction mixture contained 100 pg protein in a volume of 50 ~1. Peroxisomal fractions were treated at 0°C for 1 h with proteinase K (final concentration, 250 pg/ml), in the presence or absence of 1% (v/v) Triton X-100. Digestion was terminated by the addition of phenylmethanesulfonyl fluoride (PMSF) (final concentration, 1 mM). The samples were analyzed by native-PAGE and SDS-PAGE (10% acrylamide), followed by Western blotting. Electronmicroscopy and immunoelectronmicroscopy For electron microscopy, the cells were prefixed with 2.5% glutaraldehyde (Nisshin EM, Tokyo), and postfixed with KMn04 (26). For immunocytochemical labeling, yeast cells were fixed with a mixture of 0.5% glutaraldehyde and 3% paraformaldehyde (TA AB Laboratories Equipment, Berkshire, England) in 0.1 M potassium phosphate buffer (pH 7.6) containing 0.8% NaCl. A gold-particle-labeled goat anti-rabbit IgG was used as the second antibody (27). Native-PAGE A gradient gel which contained 424% polyacrylamide (3-15% glycerol) was used for electrophoresis in TBE buffer (89mM Tris, 89mM boric

acid, and 20 mM EDTA (pH 8.0)) for 24 h at 4°C (20 V/cm) (28, 29). After Detection of catalase activity on the gel native-PAGE, the gel was soaked for 20min in deionized water. A hydrogen peroxide solution (10%) was then poured on the gel. Bands of oxygen bubbles could be seen immediately. Western blot analysis Western blot analysis using the anti-CTC antibody was carried out as reported previously (30). Assay of catalase and protein Catalase and protein were assayed by the methods described previously (1, 31). Chemicals Restriction endonucleases were obtained from Toyobo Co. (Osaka); The DNA polymerase for PCR was obtained from Wako Pure Chemical Industries (Osaka). The electrophoresis calibration kits for SDSPAGE (94 kDa, phosphorylase b; 67 kDa, bovine serum albumin; 43 kDa, ovalbumin; 30 kDa, carbonic anhydrase; 20.1 kDa, soybean trypsin inhibitor; 14.4 kDa, alactalbumin) and for native-PAGE (660 kDa, thyroglobulin; 440 kDa, ferritin; 232 kDa bovine catalase; 140 kDa, lactate dehydrogenase; 67 kDa, bovine serum albumin) were purchased from Pharmacia (Uppsala, Sweden). RESULTS Preparation of S. cerevisiae catalase A and catalase T gene disruptants (S. cerevisiae AAAT strain) S. cerevisiae has two catalase isozymes; one is peroxisomal catalase (catalase A) and the other cytosolic catalase (catalase T). For induction of foreign catalase genes and transportation of the expressed product into peroxisomes, it is necessary to eliminate the effect of these endogenous catalases by the disruption of their genes. First, the endogenous catalase A gene (SCCA) was disrupted. The 2.8-kbp EcoRI DNA fragment encoding SCCA (20) was isolated from S. cerevisiae genomic DNA by PCR, using primers containing the SafI site and EcoRI site, namely; 5’-TAGTCGACGAAGCGTCTAG CCGATTACC-3’ and 5’-GAGAATTCGTCTCTTCCAG ATTGCTGTCG-3’. This amplified SCCA DNA fragment was introduced into the Sa&EcoRI site of pUC19 and subcloned. The subcloned plasmid DNA was digested with EcoRV to remove a 0.4 kbp fragment including the SCCA initiation codon for translation. In its stead, a 2.2-kbp fragment coding LEU2 cut off by PCR using 5’-TAGTCGACATCAGAGCAGATTGTAC-3’ and 5’GCGTCGACGCGGTATTTCACACCGCATA-3’ from pRS405 (32) and blunted with T4 DNA polymerase, was inserted into that EcoRV site and ligated. The SCCA gene disruption vector (Fig. IA-a) was digested with BamHI which was present in the multicloning site of pUC19 and in the SCCA gene. The 3.8-kbp fragment of two fragments obtained was used to transform S. cerevisiae MT8-1 to turn it to leucine prototrophs. This disruptant was named the S. cerevisiae AA strain. Disruption of the SCCA gene was confirmed by genomic Southern blot analysis of HindIll-digested DNA from a Leu+ transformant, using the BamHI-Hpal fragment of SCCA gene as the probe (Fig. lB-a). Secondly, the endogenous catalase T gene (SCCT) was disrupted. The 2.6-kbp EcoRI DNA fragment encoding SCCT (21) was isolated from S. cerevisiae genomic DNA by PCR using primers containing the BamHI site and San site, namely; 5’-ATGGATCCGCAAGTCCGAGAA

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t

TRANSPORT OF YEAST CATALASE AND HEME

LEU2 -)

.,I

573

I

a 9 .ru I

I 500b.p.

6

a

b

kbp 23.1+ *

9.46.64.4-

2.32.0-

123

4

123

4

FIG. 1. Construction of a host having no catalase activity. (A) Disruption of catalase A and catalase T genes of S. cerevisiae. (a) Disruption of catalase A gene of S. cerevisiae. The LEU2 gene, which was subcloned from pRS405 (32), was inserted into EcoRV site to disrupt catalase A gene. (b) Disruption of catalase T gene of S. cerevisiae. The HIS3 gene, which was subcloned from pRS403 (32), was inserted into the P&I-MluI gap to disrupt catalase T gene. (B) Genomic Southern blot analysis of critalase-disrupted S. cerevisiae. (a) Disruption of catalase A gene. The EarnHI-HpaI fragment (Fig. lA-a) was used as a probe. (b) Disruption of catalase T gene. The MluI-Sac1 fragment (Fig. lA-b) was used as a probe. Arrows and arrowheads represent original bands hybridized with each probe and bands corresponding to the disrupted genes, respectively, after Southern blotting. Lane 1, Genomic DNA of wild type S. cerevisiae MTS-1; lane 2, genomic DNA of S. cerevisiae AA strain; lane 3, genomic DNA of S. cerevisiae AT strain; lane 4, genomic DNA of S. cerevisiae AAAT strain.

CTAAACTG-3’ and 5’-AGGTCGACTCATGCCGATC TAAGTACCG-3’. This amplified SCCT DNA fragment was introduced into the site of pUC19 and subcloned. The amplified plasmid obtained by PCR was subcloned. Furthermore, the SalI site was changed to BumHI site by SalI digestion and ligation of a BarnHI linker. This plasmid was digested with both PstI and MluI to remove a 0.9-kbp fragment, blunted and added with a XhoI linkers on both ends. The blunted l.Zkbp of HIS3 frag-

ment from pRS403 (32) was inserted with the SalI site into the XhoI site. The SCCT gene disruption vector (Fig. IA-b) obtained after ligation was digested with BarnHI. The 2.8-kbp fragment obtained was used to transform S. cerevisiae MT8-1 and S. cerevisiae AA strain to turn them into histidine prototrophs. These disruptants were named S. cerevisiae AT and AAAT strains, respectively. Disruption of the SCCT gene was confirmed by genomic Southern blot analysis of HindIII-

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J . FERMENT.BIOENG .,

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TABLE 1.

Catalase activities in the cell-free extracts from the cells grown on YPO medium

Wild type

AA

AT (ycmolminl

1.21

0.02

AAAT

.mg protein’) 1.15

0.00

Wild type, S. cerevisiue MT&l; AA, S. cerevisiae AA strain; AT, S. cerevisiae AT strain; AAAT, S. cerevisiae AAAT strain.

digested DNA from a His+ transformant using the MU-Sac1 fragment of SCCT gene as the probe (Fig. lB-b). The catalase activities in S. cerevisiae MT8-1 (wild strain), AA, AT, and AAAT strains were measured, and are shown in Table 1. The AAAT strain had no endogenous catalase activity. There was no cross-reactive protein either in the AAAT strain or in the wild strain against anti-CTC antibody as determined by Western blot analysis. Overexpression of CTC gene in the AAAT strain under

sea I

CTC

pMT34(-G7)-ICL-CT

CTC coding region

/ NC01

UPR-ICL \ BarnHI

B

the conditions of peroxisome proliferation We demonstrated previously that catalase was able to assemble into a tetrameric form having four hemes, in the cytosol, in the absence of the proliferation of peroxisomes, using the expression system of C. tropicalis catalase (CTC) in S. cerevisiae, prepared using the GAL7 promoter (18). These results, together with the fact that cytosolic catalase was present (12, 14-17), ensure that peroxisomes themselves are not indispensable for the formation of functional catalase. However, the coordinated transport of catalase subunits and heme into the organelle has not still been clarified except that PAS10 is known to be essential (12). To elucidate the transportation of the oligomeric form of catalase into peroxisomes and the general or actual role of peroxisomes beyond yeast species in this event, the ICL promoter (19, 22) was applied to induce both catalase synthesis and peroxisome proliferation in S. cerevisiue AAAT strains growing on oleic acid. When the transformants harbouring the plasmid pMT34( - G7)-ICL-CTC (Fig. 2A) were cultivated in YPO medium, a 54 kDa protein was inducibly synthesized, to a greater extent than in YPD medium (Fig. 2B). The molecular mass and reactivity with anti-CTC antibody (Fig. 2C) revealed this protein to be a recombinant catalase (rCTC) subunit, although below the 54 kDa band, degradation products (33) were observed in the case of cells cultivated in YPO medium. rCTC in the oleic acid-grown cells was estimated to constitute about 8.8% of the soluble proteins, as estimated using the Shimadzu CS-9000 Densitometer (shown in Fig. 2B), while the catalase activity in the cell-free extract from the transformants was only 4.80 mmol/min/mg protein. Oligomeric forms of rCTC expressed in S. cerevisiue AAAT strain As mentioned above, catalase activity in the cell-free extract of S. cerevisiae AAAT strains harbouring pMT34( - G7)-ICL-CTC grown on YPO medium was much smaller than that expected from the content of the catalase subunit as estimated by SDS-PAGE, c

M(kDa) 94-

M(kDa)

’

9467f

“““V 43--

-_

30-

1

2

3

FIG. 2. Expression of rCTC in S. cerevisiae AAAT strains grown in YPO medium. (A) The plasmid pMT34(-G7)-ICL-CTC. pMT34( - G7)-ICL-CTC (9.15kbp) was constructed from the YEp-type plasmid, pMT34-( + 3) containing the GAL7 promoter, as described in Materials and Methods. Gene symbols are as follows: -G7, GAL7 omitted; Amp’, ,9-lactamase gene; 2 pm, 2 pm DNA on YEp24; CTC, C. tropicalis catalase gene; ICL, C. tropicalis isocitrate lyase gene; URA3, S. cerevisiae URA3 gene. (B) SDS-PAGE and (C) Western blot analysis of rCTC. Respective cell-free extracts (50 pg protein) were prepared from glucose-grown cells (lane 1) and oleic acid-grown cells (lane 2, 3). Lane 1, 2, Cell-free extract from the cells harbouring pMT34(-G7)-ICL-CTC. Lane 3, Cell-free extract from the cells harbouring original vector pMT34-(+3) as the control. In (B), Coomassie brilliant blue was used for staining proteins. Arrows represent the expressed rCTC subunit.

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OF YEAST

C 0.01

0

0

0.01

* k

t

‘_ : -.

:?’ 67-

575

Catalase is an enzyme with a tetrameric conformation, with one molecule of heme in each subunit, showing peroxisomal localization. The mechanism of transport of peroxisomal enzymes has been studied, with special focus on peroxisomal targeting signals (PTSs). One of the PTSs identified was C-terminal tripeptide consisting of serine-lysine-leucine (SKL) or its analogues (SKL motif) (35-37). Rat 3-ketoacyl-CoA thiolase contains another cleavable PTS located at the N-terminus (38, 39). S. cerevisiae catalase A (SCCA) has a SKL motif (SKF) (9,

M( kDa) -

AND HEME

DISCUSSION

6

669

CATALASE

20,OOOxg supernatant was 19% when the activity of acyl-CoA oxidase in the homogenate was represented as 100%). Protease treatment of the peroxisome-containing fraction revealed that rCTC was transported and localized inside peroxisomes (Fig. 4A, lane 2 and 4B, lane 3), since the protein in intact peroxisomes was resistant to the protease. Peroxisomal localization of a part of rCTC in S. cerevisiae AAAT strains was also confirmed by immunocytochemical observations (Fig. 5). Overexpressed proteins were localized in the cytoplasm. The results shown in Fig. 4B indicated that a part of the rCTC recovered in the peroxisome-containing fraction (about 20%) was actually localized inside peroxisomes, and the rest (about 80%) was strongly associated on peroxisomes. In peroxisomes of C. tropicalis, CTC is present only as the tetrameric form (34). It is important to clarify whether or not all of the rCTC transported inside peroxisomes of S. cerevisiae assemble into the tetrameric form (220 kDa) and has the activity as in the case of C. tropicalis. Western blot analysis after native-PAGE (Fig. 4A) suggested the possibility that all of the rCTC transported inside peroxisomes of S. cerevisiae was probably only the tetrameric form and multimeric forms, except for the tetrameric form, associated on the outside of the peroxisomai membrane couId not be transported inside peroxisomes .

although a catalase activity of 14.1 mmol/min/mg protein must be obtained from calculations based on the activity of the purified catalase (18). To clarify this discrepancy, the cell-free extract was subjected to nativePAGE, followed by Western blot analysis using the antiCTC antibody (Fig. 3A). Several multimeric forms larger than a tetrameric form (220 kDa) of CTC from alkanegrown C. tropicalis (lane 2), and rCTC purified from S. cerevisiae (lane 3), were observed in the transformants (lanes 1 and 4). Smaller forms, below the 220 kDa band, probably represented dimers or monomers. To examine whether or not all of these multimeric forms possess catalase activity or not, the detection of the catalase activity was carried out (Fig. 3B). The most part of the activity was detected for the 220 kDa tetrameric form, although this tetrameric form shared only 18.5%, on Western blot analysis (Shimadzu CS-9000 Densitometer), of the total amount of multimers expressed (in Fig. 3A, lane 1). Since the holo-enzyme of rCTC having heme had an absorption maximum at 406nm, the gel was scanned at 406 nm. Obvious absorption was observed only for the tetrameric form (220 kDa), as shown in Fig. 3C. The monomeric form, defective in heme, seemed to aggregate to form dimers or multimers. Similar results were obtained with the cell-free extract from the pMT34-CTCharbouring S. cerevisiae (18), overexpressing the catalase subunit (Fig. 3A, lane 4). Subcellular localization of rCTC in S. cerevisiue MAT strain To investigate the peroxisomal localization of rCTC in S. cerevisiae under conditions of peroxisome proliferation, subcellular fractionation was carried out. Since it was difficult to isolate only peroxisomes from S. cerevisiae AAAT strains because of their fragility, the particulate fraction obtained by centrifugation at 20,OOOxg after homogenization of protoplasts was used as the peroxisome-containing fraction, judging from the recoverv of the other neroxisomal marker enzvme. acvl-CoA oxidase (the recovery of acyl-CoA oxidase ;n the 20,000 xg particulate fraction was 81% and that in the

A

TRANSPORT

~ 1

FIG. 3. Characterization of rCTC expressed in oleic acid-grown S. cerevisiue AAAT strains. (A) Various forms of rCTC in native-PAGE. Respective cell-free extracts (50 pg protein) prepared from oleic acid-grown S. cerevisiue AAAT strains harbouring pMT34( - G7)-ICL-CTC (lane l), alkane-grown C. tropicalis (lane 2), galactose-grown S. cerevisiae harbouring pMT34-CTC (lane 4), and 1 ,ng of purified rCTC (lane 3) (18). Anti-CTC antibody was used (lanes 1, 2, and 4). Lane 3 was stained with Coomassie brilliant blue. (B) Detection of catalase activity of and (C) scanning for absorption of heme at 406 nm (Shimadzu CS-9000 densitometer) of the cell-free extracts (50 pg protein) from the oleic acid-grown AAAT cells harbouring pMT34(-G7)-ICL-CTC (lane 1) and pMT34( +3) as a control (lane 2). Arrows represent the 220 kDa-tetrameric form of rCTC.

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J. FERMENT. BIOENG.,

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M(kDa) 669-

4-

232-

671

2

M(kDa) 67-

30-

1234 FIG. 4. Protease treatment of the peroxisome-containing fraction of oleic acid-grown S. cerevisiae AAAT strains. (A) Native-PAGE (each, 100,ng protein): lane 1, no treatment; lane 2, treatment with proteinase K. The arrow represents the 220 kDa-tetrameric form of rCTC. (B) SDS-PAGE (each, 1OO~g protein): lane 1, no treatment; lane 2, treatment with 1% Triton X-100; lane 3, treatment with proteinase K; lane 4, treatment with the detergents and proteinase K. The arrow represents the 54 kDa subunit of rCTC. After electrophoresis, Western blot analysis was done using the anti-CTC antibody.

10, 20),but C. tropicalis catalase (CTC), which exhibited 38.8% similarity to SCCA, does not have the SKL motif, the C-terminal tripeptide being PRK (40). However, it was demonstrated that C. tropicalis catalase produced in S. cerevisiae, whether the tetrameric form or multimeric forms, could translocate to the peroxisomal surface of S. cerevisiae AAAT strains (Fig. 4). These results suggest that a common signal derived from the primary amino acid sequence between CTC and SCCA should be present besides the C-terminal region. Meanwhile, binding with heme and assembly into the tetrameric form of catalase subunits remain obscure. One catalase subunit is able to bind one molecule of heme at several transport stages and in different states of assembly; in cytosol before catalase reaches the peroxisomal membrane or in peroxisomal matrix after being transported (13), and as a monomer or a multimer. Although it has been predicted that a translated monomer translocates to peroxisomes and bind with

FIG. 5. Electron micrographs of S. cerevisiae AAAT cells grown on YPO medium fixed with KMn04 (a, c) and immunoelectronmicrographs (b, d) with anti-CTC antibody. S. cerevisiae AAAT strains harbouring pMT34-(+3) (a, b) and pMT34(-G7)-ICL-CTC (c, d), were used respectively. In a small panel in d, the peroxisome-containing fraction obtained by centrifugation at 20,000 x g was shown to clearly demonstrate the peroxisomal membrane, because peroxisomal membrane was difficult to identify in S. cerevbiae AAAT cells grown on YPO medium, although peroxisomes were represented with dotted lines. M, Mitochondrion; N, nucleus; P, peroxisome; V, vacuole.

heme in them (3, 13), a new hypothesis has been presented recently (16, 41, 42), that is, catalase in human skin fibroblasts assembles into a tetrameric conformation in the cytosol once, and then unfolding of the tetrameric form occurs prior to its transport to peroxisomes. However, details of the mechanism, such as binding of heme with catalase subunits during transportation, are not clear. From the results shown in Figs. 3 and 4, the possibility is suggested that almost all the rCTC transported into the peroxisomes of S. cerevisiae assembled as the tetramerit form, while it formed various multimers on the surface of peroxisomes, which were inactive except for the tetrameric form. The presence of some novel mechanism in the transportation of oligomeric proteins accompanying with cofactors into peroxisomes has been expected. Furthermore, these phenomena seem to suggest the possibility that peroxisomes selectively transport the rCTC subunit and heme at a ratio of 1 : 1 or their active and tetrameric assembly form, together with the result demonstrated on C. tropicalis peroxisomal catalase (34), in which no excess of free heme was present in peroxisomes. Therefore, it would be of interest to investigate the transport mechanism of the oligomeric form of catalase into peroxisomes after the recognition of peroxi-

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somal membranes by catalase and its association with the membrane by focusing on the behavior of heme. Further studies with rCTC mutants, which cannot bind with heme or make a tetrameric form, and using a specific catalase inhibitor will offer promise for better understanding of the mechanism. REFERENCES 1. Kawamoto, S., Tanaka, Fukui,

S.,

and

Osumi,

A., Yamamura, M.: Microbody

yeast. Enzyme localization Microbial., 112, 1-8 (1977).

M.,

Teranishi,

Y.,

of n-alkane-grown in the isolated microbody. Arch.

A., Osumi, M., and Fukui, S.: Peroxisome of alkanegrown yeast: fundamental and practical aspects. Ann. N.Y. Acad. Sci., 386, 183-199 (1982). 3. Lazarow, P. B. and Fujiki, Y.: Biogenesis of peroxisome. Ann. Rev. Cell Biol., 1, 489-530 (1985).

2. Tanaka,

4. McCammon, M. T., Veenhuis, M., Trapp, S. B., and Goodman, J. M.: Association of glyoxylate and beta-oxidation enzymes

with peroxisomes of Succharomyces cerevisiae. J. Bacterial., 172, 5816-5827 (1990). 5. Kamiryo, T., Abe, M., Okazaki, K., Kate, S., and Sbimamoto, N.: Absence of DNA in peroxisomes of Cundida tropicalis. J. Bacterial., 152, 269-274 (1982). 6. Fukuda, Y., Atomi, H., Kuribara, T., Hikida, M., Ueda, M., and Tanaka, A.: Genes encoding peroxisomal enzymes are not

necessarily assigned on the same chromosome of an n-alkaneutilizable yeast Candida tropic&s. FEBS Lett., 286, 61-63 (1991). 7. Goldman, B. M. and Blobel, G.: Biogenesis of peroxisomes: intracellular site of synthesis of catalase and &case. Proc. Natl. Acad. Sci. USA. 75. 5066-5070 t1978). P. B.: Post-t&slational import of 8. Fujiki, Y. and Lazaiow; fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro. J. Biol. Chem., 260, 5603-5609 (1985). 9. Hartig, A., Ogris, M., Cohen, G., and Binder, M.: Fate of

highly expressed proteins destined to peroxisomes in Saccharomyces cerevisiae. Curr. Genet., 18, 23-27 (1990). 10 Kragler, F., Langeder, A., Raupachova, J., Binder, M., and Hartig, A.: Two independent peroxisomal targeting signals in catalase A of Saccharomyces cerevisiae. J. Cell Biol., 120, 665673 (1993). 11. Didon, T. and Roggeukamp, R.: Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansen&a polymorpha. FEBS Lett., 303, 113-116 (1992). 12. van der Leu, I., Franse, M. M., Elgersma, Y., Distel, B., and Tabak, H. F.: PAS10 is a tetratricopeptide-repeat protein that is essential for the import of most matrix proteins into peroxisomes of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, 90, 11782-11786 (1993). 13. Lazarow, P. and Duve, C.: The synthesis and turnover of rat liver peroxisomes. V. Intracellular pathway of catalase synthesis. J. Cell BioI., 59, 507-524 (1973). 14. Seah, T. C. M. and Kaplan, J. G.: Purification and properties of the catalase of baker’s yeast. J. Biol. Chem., 248, 28892893 (1973). 15. Yamamoto, K., Volkl, A., Hashimolo, T., and Fahimi, H. D.: Catalase in guinea pig hepatocytes is localized in cytoplasm, nuclear matrix and peroxisomes. Eur. J. Cell Biol., 46, 129135 (1988). 16. Wanders, R. J. A., Strijland, A., Roermund, C. W. T., Bosch, H., Schutgens, R. B. H., Tager, J. M., and Scbram, A.: Catalase in cultured skin fibroblasts from patients with the cerebrohepato-renal (Zellweger) syndrome; normal maturation in peroxisome-deficient cells. Biochim. Biophys. Acta, 923, 478482 (1987). 17. Ceerts, A., Prest, B. D., and Roels, F.: On the topology of the catalase biosynthesis and -degradation in the guinea pig liver. Histochem., 80, 339-345 (1984). 18. Kinoshita, H., Atomi, H., Ueda, M., and Tanaka, A.: Characterization of the catalase of the n-alkane-utilizing yeast Candida

TRANSPORT OF YEAST CATALASE AND HEME

577

tropicalis functionally expressed in Saccharomyces cerevisiae. Appl. Microbial. Biotechnol., 40, 682-686 (1994). 19. Umemura, K., Atomi, H., Kanai, T., Teranisbi, Y., Ueda, M., and Tanaka, A.: A novel promoter, derive from the isocitrate lyase gene of Cundida tropicalis, inducible with acetate in Saccharomyces cerevisiue. Appl. Microbial. Biotechnol., 43, 489492 (1995). 20. Cohen, G., Rapatz, W., and Ruis, H.: Sequence of the Saccharomyces cerevisiae CTAl gene and amino acid sequence of catalase A derived from it. Eur. J. Biochem., 176, 159-163 (1988). 21. Hartig, A. and Ruis, H.: Nucleotide sequence of the Succhuromyces cerevisiae CTTl gene and deduced amino-acid sequence of yeast catalase T. Eur. J. Biochem., 160, 487-490 (1986). 22. Atomi, H., Umemura, K., Higasbijima, T.. Kanai, T., Yotsumoto, Y., Teranishi, Y., Ueda, M., and Tanaka, A.: The upstream region of the isocitrate lyase gene (UPR-ICL) of Candida tropicalis induces gene expression in both Saccharomyces cerevisiae and Escherichia coli by acetate via two distinct promoters. Arch. Microbial., 163, 322-328 (1995). 23. Tajima, M., Nogi, Y., and Fukasawa, T.: Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast, 1, 67-77 (1985). 24. Ito, H., Fukuda, Y., Murata, K., and Kimura, A.: Transformation of intact yeast cells treated

with alkali

cations.

J. Bac-

teriol., 153, 163-168 (1983). 25. Atomi, H., Ueda, M., Suzuki, J., Kamada, Y., and Tanaka, A.: Presence of carnitine acetyltransferase in mitochondria of oleic acid-grown Saccharomyces cerevisiae. FEMS Microbial. Lett., 112, 31-34 (1993). 26. Numata, K., Ueki, T., Naito, N., Yamada, N., Kamasawa, N., Oki, T., and Osumi, M.: Morphological changes of Candida albicans induced by BMY-28864, a highly water-soluble pradimicin derivative. J. Electron Microsc., 42, 147-155 (1993). 27. Kamasawa, N., Ohtsuka, I., Kamada, Y., Ueda, M., Tanaka, A., and Osumi, M.: lmmunoelectron microscopic observation of the behaviors of peroxisomal enzymes inducibly synthesized in an n-alkane-utilizable yeast cell, Candidu tropicalis. Cell Struct. Funct., 21, 117-122 (1996). 28. Andersson, L. O., Borg, H., and Mikaelsson, M.: Molecular weight estimations of proteins by electrophoresis in polyacrylamide gels of graded porosity. FEBS Lett., 20, 199-202 (1972). 29. Clos, J., Westwood, J. T., Becker, P. B., Wilson, S., Lambert, K., and Wu, C.: Molecular cloning and expression of a hexamerit Drosophila heat shock factor subject to negative regulation. Cell, 63. 1085-1097 (19901. 30. Ueda, M., Mozaffar, S., Atodi, H., Osumi, M., and Tanaka, A.: Characterization of peroxisomes in an n-alkane-utilizable yeast, Candida tropicab, grown on glucose and propionate. J. Ferment. Bioeng., 68, 411-416 (1989). 31. Kuribara, T., Ueda, M., Okada, H., Kamasawa, N., Naito, N., Osumi, M., and Tanaka, A.: /?-Oxidation of butyrate, the short chain length fatty acid, occurs in peroxisomes in the yeast, Candida tropicalis. J. Biochem., 111, 783-787 (1992). 32. Sikorski, R. S. and Hieter, P.: A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics, 122, 19-27 (1989). 33. Okamoto, H., Ueda, M., Yamanoi, K., Ukita, R., Naito, N., Osumi, M., and Tanaka, A.: Degradation of peroxisomal catalase in an n-alkane-utilizable yeast, Candida tropicalis. J. Ferment. Bioeng., 72, 254-257 (1991). 34. Soga, O., Kinoshita, H., Ueda, M., and Tanaka, A.: Evaluation of peroxisomal heme in yeast. J. Biochem., 121, 25-28 (1997). 35. Gould, S. J., Keller, G. A., and Subramani, S.: Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J. Cell Biol.. 105. 2923-2931 (1987). 36. Gould, S. J., Keller, G. A., and Suhramani, s.: Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J. Cell Biol., 107, 897-905 (1988).

578

KINOSHITA ET AL.

37. Gould, S. J., Keller, G. A., Hosken, N., Wilkinson, J., and Subramani, S.: A conserved tripeptide sorts proteins to peroxisomes. J. Cell Biol., 108, 1657-1664 (1989). 38. Swiokels, B. W., Gould, S. J., Bodnar, A. G., Rachubioski, R. A., and Subramani, S.: A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. EMBO J., 10, 3255-3262 (1991). 39. Osumi, T., Tsukamoto, T., Hata, S., Yokota, S., Miura, S., Fujiki, Y., Hijikata, M., Miyazawa, S., and Hasbimoto, T.: Amino-terminal presequence of the precursor of peroxisomal 3ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem. Biophys. Res. Commun., 181, 947954 (1991).

J. FERMENT.BIOENG., 40. Okada, H., Ueda, M., Sugaya, T., Atomi, H., Momffar, S.,

Hishida, T., Teranishi, Y., Okazaki, K., Take&i, T., Kamiryo, T., and Tanaka, A.: Catalase gene of the yeast Candida tropicalis: sequence analysis and comparison with peroxisomal and cytosolic catalases from other sources. Eur. J. Biochem., 170, 105-110 (1987). 41. Middelkoop, E., Strijiland, A., and Tager, J. M.: Does aminotriazole inhibit import of catalase into peroxisomes by retarding unfolding? FEBS Lett., 279, 79-82 (1991). 42. Middelkoop, E., Wiemer, E. A. C., Shoenmaker, D. E. T., Strijland, A., and Tager, J. M.: Topology of catalase assembly in human skin fibroblasts. Biochim. Biophys. Acta, 1220, 15% 20 (1993).