Eur J. Biochem IK4, 173-179 (1989) 8.) FEBS 1989

Functional complementation of catalase-defective peroxisomes in a methylotrophic yeast by import of the catalase A from Saccharomyces cerevisiae Hans HANSEN and Rainer ROGGENKAMP Institut fur Mikrobiologie der Heinrich-Heine-Univcrsitlt Dusseldorf

(Received March 17/June 6, 1989) - EJB 89 0326

A mutant of the methanol-utilizing yeast Hansenula polymorphu defective in catalase was isolated. It lacks the ability to grow on methanol as the sole source of carbon and energy due to a loss of peroxisomal function that is required for the dissimilation and assimilation of this substrate. Growth of the mutant on glucose or glycerol was not impaired. Transformation of mutant cells with the gene coding for catalase A from Saccharomyces cerevisiae [Cohen, G., Fessl, F., Traczyk, J., Rytka, J. & Ruis, H. (1985) Mol. Gen. Genet. 200, 74-79] conferred constitutive expression of catalase activity. When the gene was placed under control of the regulatory methanol oxidase promoter from H . polymorpha, high levels of activity subject to glucose repression were obtained. In both cases efficient targeting of catalase A to the heterologous peroxisomes and assembly into an active form could be demonstrated. Concomitantly, growth on methanol was restored in the transformed mutant. The results are in line with a high conservation of transport signals on peroxisomal proteins. Expression of a cytosolic catalase in H. polymorpha did not confer the ability to grow on methanol. Therefore, proper localization of the catalase activity is a prerequisite for peroxisomal function.

The term ‘microbody’ defines cellular organelles (peroxi- somes, glyoxysomes) that are ubiquitous in eukaryotic cells. According to cell type or metabolic state of a cell, they carry out diverse metabolic functions; their synthesis often appears to be inducible [l -41.

Proteins destined for microbodies should harbor certain sequence information that ensures import, i.e. sorting and membrane translocation (for a review see [5]). The diversity of microbodies also poses the question of whether protein targeting underlies a common mechanism or not. This can be answered by heterologous expression of peroxisomal proteins. Methylotrophic yeasts and Saccharomyces cerevisiae are suit- able for such studies since both yeast types contain numerous inducible peroxisomes of different size and morphology [4, 61 and of different metabolic functions. Whereas peroxisomes in methylotrophic yeasts harbor dissimilatory and assimilatory enzymes of methanol utilization [4, 7-91, in S. cerevisiue enzymes involved in p-oxidation of long-chain fatty acids are peroxisomal [lo, 111. As common for all peroxisomes an intrinsic catalase is present at high amounts in both organisms [6] offering the possibility to assess heterologous transport in combination with functional complementation.

The present studies describe the isolation of a catalase mutant -in Hansenula polymorpha that abolishes peroxisome function and thus the capability to grow on methanol. After

Correspondence to R. Roggenkamp, Institut fur Mikrobiologie, Heinrich-Heine-Universitat Diisseldorf, GebHude 26.1 2, Universitats- strasse 1, D-4000 Dusseldorf, Federal Republic of Germany

Abbreviations. YEPD, 1 % yeast extract, 2% peptone, 2% glu- cose; YNB, yeast nitrogen base; ABTS, 2,2’-di-azino-(3-ethyl- beiizthiazolinsulfonale-6).

Enzymes. Catalase or hydrogcn peroxide oxidoreductase (EC 1.11.1.6); methanol oxidasc or alcohol oxidase (EC 1.1.3.13); orotidine-5’-phosphate decarboxylase (EC 4.1 .I .23); malate dehydro- genase (EC 1.1.1.37).

transformation of mutant cells with the catalase A gene from S. cerevisiae, we can demonstrate the occurrence of heterologous transport by measuring catalase activity in iso- lated peroxisomes. Furthermore, we show that the catalase A activity restores peroxisomal function in mutant cells of H. polymorpha.

MATERIALS AND METHODS Strains and media

Escherichia coli MB 1000 (hsrK hsmK lac trp pyrF) was used as bacterial host for plasmid amplification [12]. It was grown either in LB or M9 medium [13] supplemented with 100 kg ampicillin/ml and 20 pg tryptophan/ml, respectively. A thermophilic, homothallic strain (ATCC 34438) was used as a wild-type strain of Hansenula polymorpha. The mutant MCT-75 (odcl ca t l ) lacks orotidine-5‘-phosphate decar- boxylase and catalase activity. Complex medium for yeast was YEPD. Minimal medium was 0.67% YNB without amino acids supplemented with carbon sources as indicated and, if necessary, with 50 pg uracil/ml.

Plasm id5 Plasmid pHCA-159 (Fig. 1) was constructed by inserting

a 4.4-kbp Clal - SphI fragment, which carries the catalase A gene of S. cerevisiae including 1.2 kbp of the 5’-noncoding region within plasmid YEpl3-CTA1-I [14] into the corre- sponding sites of the H . polymorpha vector pHARSl [15]. Plasmid pHCT-124 (Fig. 1) carries a DNA fragment which contains the catalase T structural gene of S. cerevisiue includ- ing 0.9 kbp of the 5‘-noncoding region. This was accomplished by replacing a BamHI - PvuI fragment of pHARS1 with a 2.9-kbp BamHI - PvuI fragment of plasmid YEp13-73 [16].

174

1 S

S

W B g )

Fig. 1. Pkurrnid consrrucrions. MP, methanol oxidase promoter; MT, methanol oxidase transcription terminator. B, BamHI; Bg, Bg/II; Bs, BstEII; C , CIuI; E, EcoRI; H, HindIII; P, Pvul; S, ScaI; Sp, SphI. See tcxt for further details

Plasmid pMCA-231 was constructed by insertion of a 3.5- kb ScaI fragment from YEpl3-CTA1-I [14] containing the catalase A gene from S . cereiisiae (lacking its own promoter) into the filled up EcoRI and BglII sites of pMEX (Fig. 1). Plasmid pM EX was originally constructed for the expression of the bacterial blu gene in H . polymorpha using the methanol oxidase promoter and terminator elements (M. Eckart and C. P. Hollenberg, unpublished results).

Eiizymes uizd chemicals Restriction enzymes as well as T4 ligase and ABTS were

obtained from Soehringer (Mannheim, FRG) and ethyl methanesulfonate from Sigma (St Louis, USA). Peroxidase

was purchased from Serva (Heidelberg, FRG). Zymolyase was from Miles Scientific (Naperville, IL, USA). Chemicals used for culture media were obtained either from Difco (Detroit, MI, USA) (YNB) or from Gibco/BRL (Karlsruhe, FRG). [ U - ~ ~ P I ~ A T P and alkaline phosphatase conjugated with F(ab'), fragments of goat anti-(rabbit IgG) (for immuno- blots) were purchased from Amersham-Buchler (Braun- schweig, FRG). All other chemicals were of analytical grade available from commercial sources.

Isolation of catalase mutants H . polymorpha was grown at 37°C in 3 ml YEPD until

late-exponential growth phase. Cells were washed once in 0.1 M sodium phosphate pH 8.0 and suspended in 3 ml of the same buffer. Ethyl methanesulfonate was added to a final concentration of 3% (by vol.). The solution was vigorously mixed and incubated at 30°C until 80% of the cells had been killed. The reaction was stopped by a brief centrifugation and immediate washing of the cells with 6% (massivoi.) sodium thiosulfate and further incubation in the same solution for 10 min at room temperature. Subsequently, the cells were incubated for 48 h in 2 ml YEPD and, after growth for 24 h, replica-plated on YNB plates supplemented with 1% meth- anol. Colonies that were unable to grow on methanol as a carbon source were screened for a catalase-negative pheno- type by measuring catalase activity in crude extracts.

Transformation of yeast cells

with poly(ethy1ene glycol) as described earlier [15]. H . polymorpha was transformed using frozen cells treated

Isolation of peroxisomes A procedure that had been developed for the isolation of

peroxisomes in the methylothrophic yeast Cundidu hoidinii [8, 91 was employed with modifications. Yeast cells were grown until mid-exponential growth phase and harvested by centrifugation. The cells were washed in 50 mM potassium phosphate pH 7.5 containing 1.2 M sorbitol, resuspended in the same solution supplemented with 100 mM mercaptoetha- no1 and incubated at 30°C for 20 min. After centrifugation, yeast cells were converted to protoplasts in 50 mM potassium phosphate pH 7.5, + Zyniolyase (2 mg/g wet cells). Incu- bation was monitored for 90min at 30°C with continous pumping of the cell suspension through a perforation of 0.5 mm diameter to prevent clotting until 80% of the cells were converted to protoplasts. All subsequent steps were car- ried out at 4°C unless otherwise stated. Protoplasts were washed gently with 5 mM Mes pH 6.0,l M sorbitol and lysed in the same buffer by lowering the sorbitol concentration to 0.5 M. After 2 h of lysis the sorbitol concentration wits raised again to 1 M. The solution was centrifuged at 500 x g in order to remove whole cells and then at 17000 x g. The pellet was carefully resuspended in 5 mM Mes pH 6.0, 1 M sorbitol and applied to a sucrose gradient consisting of 5% steps in the range of 30-60% (by mass) sucrose in 5 mM Mes pH 6.0. Gradients were centrifuged in a swinging bucket rotor (SW 28, Beckman Instruments) for 4.5 h at 27000 rpm. Tubes were punctured at the bottom and about 25 1.5-mi fractions were collected.

Analytical methods Catalase activity was measured spectrophotometrically at

240 nm by H202 degradation [17], methanol oxidase by oxi-

175

dation of ABTS at 420 nm [18] and malate dehydrogenase by oxidation of NADH with oxaloacetate as a substrate 1191. Protein was determined by the Lowry procedure [20]. Yeast minipreparations were done according to Struhl et al. [21]. Nick translation and Southern analysis were performed as described by Maniatis et al. [13].

Purification of catalase for the production of specific untihodies

H . polymorpha cells were grown in YNB + 1% methanol, washed once in 100 mM Tris/HCl pH 7.3 and resuspended in the same buffer (2 ml bufferlg wet cells). Crude extracts were obtained with a Braun homogenizer (Braun, Melsungen, FRG) using glass beads (0.4--0.5 mm in diameter). Cell debris were removed by centrifugation at 20000 x g. The supernatant was dialyzed overnight against 20 mM Tris/HCl pH 7.3 and applied to a DEAE-Sephadex (Pharmacia) column (2.5 x 25 cm). Proteins were eluted with a gradient of 400 ml 20 mM Tris/HCl and 400 ml 300 mM Tris/HCl pH 7.3; 80 fractions of about 10 ml each were collected. Fractions 36- 41, containing the highest catalase activities, were pooled and concentrated to 1 ml by dialysis against a solution of 20% (mass/vol.) poly(ethy1ene glycol) 20000 in 50 mM Tris/HCl pH 7.0. Proteins were further separated on a SephacryLS300 (Pharmacia) column (1.5 x 80 cm) with a flow rate of 4 ml/h and a fraction size of 0.8 ml. A total of 25 fractions exhibiting the highest catalase activities were pooled, concentrated with poly(ethy1ene glycol) as described above and frozen at - 20°C for further use.

For immunization of rabbits, aliquots of the protein were electrophoresed on 8% polyacrylamide gels in the presence of SDS [22]. The catalase band of about 60 kDa was visualized by soaking the gel in ice-cold 0.1 M KCI, cut out and dialyzed against 50 mM sodium phosphate pH 7.5 containing 0.1% SDS, for 48 h at 37°C. The protein was concentrated by freeze-drying and antibodies were raised in rabbits using Freunds adjuvants (Difco) as described earlier 1231. Immuno- blots of electrophoresed proteins were performed as reported 1241.

RESULTS

Isolation q j cutaluse mutants

During growth of H. polyrnorpha on methanol the oxi- dation of one molecule methanol via methanol oxidase in peroxisomes leads to the generation of one molecule hydrogen peroxide. Therefore, we reasoned that catalase mutants would disrupt peroxisomal function and consequently loose the ability to grow on methanol as a sole carbon and energy source. One catalase mutant, designated M-75 (catl) , was detected among a total of 60 mutants that were unable to grow on methanol. Enzymatic activity of catalase was undetectable in this mutant (see Table 1), but inactive protein (at reduced quantities) could be identified immunologically (see Fig. 3). N o revertants of M-75 were found when 10’ cells were plated. Only a few pseudorevertants that formed minicolonies were detected after four weeks on methanol plates.

Mutant cells were again mutagenised and uracil auxotrophs lacking orotidine-5’-phosphate decarboxyhse were selected by 5’-fluoro-orotic acid [I 51. The resulting double mutant MCT-75 (cut1 odcl) was used for all trans- formations with catalase genes using the URA3 gene of S. cerevisiue as a selection marker 1151.

Table 1. Specific uctivities of cutalases in crude extracts of’ H. poly- morpha transfirmanrs Cells were grown in yeast nitrogen base + 1 YO of the carbon source. Plasmid pMCA-231 was cut with Hind111 before transformation. A unit (U) of activity is thc amount catalyzing the degradation of 1 pmol H202/min

Strain/plasmid Specific activity of catalasc in cells grown on

glucose glycerol methanol

U/mg protein

Wild-type 80 780 1900 MCT-75 0 0 0 MCT-75/pHCA-I 59 270 250 260 MCT-75/pHCT-124 60 50 890 MCT-75/pMCA-231 5 1430 3200

Fig. 2. Southern analysis of plasmids pHCA-159 and PHCT-124 in transformants of MCT-75. Lane 1, pHCA-159 digested with EcoRI (control); lane 2, autonomously replicating plasmid pHCA-2 59, undigested; lane 3, as lane 2 but digested with EcoRT; lane 4, oligomerized copies of plasmid pHCA-159, undigested; lane 5 , as lane 4, but digcsted with EciRI ; lancs 6- 10, plasmid pHCT-124, analysed in thc same way as dcscribcd for lanes 1-5, reading in the opposite direction. Arrow shows the position of chromosomal DNA, undigested. Lanes I - 5 were probed with nick-translated plasmid pIlCA-159 and lanes 6- 10 with nick-translated pHCT-I24

Transformation c?f’ mutant cells with catalase genes of S. cerevisiae

Both the peroxisomal catalase A and the cytoplasmic catalase T of S. cerevisiue were expressed in H. polyrnorpha. Mutant MCT-75 was transformed to uracil prototrophy with vectors pHCA-159 and pHCT-124. Transformations were confirmed by Southern analysis showing the autonomously replicating plasmids in yeast minilysates (Fig. 2, lanes 2 and 9). Catalase activities were detected in both transformants. Non-selective growth resulted in a concomitant loss of enzyme activity and uracil prototrophy demonstrating plasmid-me- diated expression. Specific catalase activities were low in com- parison to wild-type levels, obviously due to a poor recog-

176

Fig. 3. Immunological detection of catalases by Western blot hybridiza- tion in crude extracts of'MCT-75 transformed with pHCA-159,pHCT- 124 und pMC'A-231. Molecular mass markers in kDa (from top to bottom), phosphorylase b, bovine albumin, egg albumin. wt, wild- type; cHp, wild-type catalase; cT, catalase T; cA, catalase A. Carbon sources used: g, glucose; y, glycerol; m, methanol. Aliquots containing 200pg protein were loaded on each slot. Antisera against H. polyrnorphu catalase and crude extracts were obtained as described in Materials and Methods

nition of the heterologous promoters in H . polymorpha and the great instability of autonomously replicating plasmids [15]. However, upon prolonged growth on selective medium, catalase activities increased steadily along with the number of colonies that appeared as stable uracil prototrophs. Southern analysis of such a stable prototroph demonstrated the occur- rence of plasmid oligomerization (Fig. 2, lanes 4, 5 and 6, 7). Thereby, the specific enzyme activity reached 200 - 300 U/ mg protein in the case of catalase A (Table 1). Catalase T transformants exhibited a specific activity of about 50 U/ mg protein (Table 1). Although both the two catalases in S. cerevisiae and the catalase in H. polymorpha are subject to catabolite repression, no significant regulation was observed in the transformants by comparing activities after growth on glucose and the non-repressible carbon source glycerol (Table 1). Thus either the heterologous catalase promoters are not recognized in H . polymorpha by a regulatory element or such an element is titrated out because of the high copy number expression. The second possibility, however, is not likely, since overexpression of the peroxisomal methanol oxidase in H. polymorpha has retained regulation by glucose [24].

Immunological analysis qf catalases

Since enzymatic activities both of catalase A and catalase T expressed in H. polymorpha showed a reduced stability in crude extracts in comparison to the host enzyme (data not shown), i t was necessary to substantiate the activity data (Table 1) by quantification of the proteins. Thereby we made use of the fact that antibodies generated against the H . polynwrpha catalase cross-reacted with both enzymes from S. cere visiue.

Crude extracts of both transformants were subjected to polyacrylainide gel electrophoresis in the presence of SDS and subsequently analysed by immuno-blots on nitrocellulose. Catalase A was identified as a protein band of lower molecular mass than the H . polymorpha catalase (Fig. 3). Since the catalase mutant MCT-75 of H . polymorpha accumulates an inactive catalase, in the transformants a double band of catalase A and the H . polymorpha catalase appeared (Fig. 3).

A comparison of transformed cells grown either on glycerol or glucose clearly indicated that the mutant catalase, though present at reduced amounts, had retained regulation by glu- cose repression like the wild-type enzyme. In contrast, the intensity of immuno-signals of catalase A was comparable when transformed cells were grown under either repressing or derepressing conditions (Fig. 3, pHCA-159). With respect to protein amount and regulation of catalase A, the immunological data confirmed the activity data presented in Table 1. Immuno-blot analysis of catalase T in H . polymorpha gave rise to protein bands with comparable staining intensities at the detection limit in accordance with the low specific activity (Table 1) under repressing or depressing conditions (Fig. 3, pHCT-124). On the other hand, methanol-grown cells exhibited a strong signal of catalase T (Fig. 3, pHCT-124) which is in line with a severalfold increase in specific activity (Table 1).

The different electrophoretic mobilities of the catalases A and T by SDSjPAGE (Fig. 3) are in agreement with their molecular masses of 58.6 and 64.5 kDa according to the nucleotide sequence data of the corresponding genes [25, 261.

Catalase A activity in isolated peroxisomes of transformants

Protoplasts of transformed yeast cells were subjected to cell fractionation. Organelle pellets of catalase A transform- ants contained significant amounts of enzyme activity. Only traces of activity could be sedimented in the case of catalase T in line with its cytoplasmic nature. The results suggested import of catalase A into the heterologous peroxisomes of H . polymorpha. This was substantiated by isolation of peroxisomes on a sucrose gradient and enzyme activity mea- surements. Catalase activity was found to be associated with a fraction migrating at a lower position than mitochondria and cosedimenting with methanol oxidase as a peroxisomal marker (Fig. 4A). Since a 15000 x g pellet was loaded on the sucrose gradient (see Materials and Methods), the smaller activity peak of malate dehydrogenase as a mitochondria1 marker enzyme on top of the gradient may reflect a certain leakage of mitochondria rather than activity of the cytosolic malate dehydrogenase. As expected, no association with any particular organelle fraction in the gradient of catalase T was observed (Fig. 4B). We conclude that topogenic signals on catalase A confer transport into peroxisomes of H. polymorpha followed by assembly to an active enzyme con- figuration.

Complementation of peroxisome function by catalase A

The catalase A activity in peroxisomes of transformed cells was tested with respect to methanol utilization. Growth on methanol as the sole source of carbon and energy was monitored. Remarkable growth rates of catalase A transrorm- ants were obtained reaching stationary growth phase after 3-4 days of cultivation (Fig. 5) . N o significant growth on non-transformed cells or catalase T transformants was ob- served within this time period.

Since catalase activity could be measured with whole H . polymorphu cells (data not shown), hydrogen peroxide was able to leave the peroxisomes by diffusion through the peroxisomal membrane that was also shown to be permeable for compounds of low molecular mass in rat liver peroxisomes [27]. Therefore, one could expect that the cytoplasmic catalase T activity would suffice to restore growth on methanol. In fact, extended incubation periods resulted in very slow growth

177

m a, U

0

8

6

4

2

5 10 15 20 Fraction

Fig. 4. Separation of' peroxisomes in sucrose gradients isolated from MCT-75 cells transformed with pHCA-IS9 andpHCT-124. Cells were grown on 3% glycerol plus 0.5'1/0 methanol up to late-logarithmic phase. Sucrose concentration was 30-60%; 23 fractions of about 1.7 ml were collected and relative activities of methanol oxidase (0), catalase ( 0 ) and malate dehydrogenase (0) were determined. The latter enzyme was used as a mitochondria1 marker. (A) pHCA-159; (B) pHCT-124

1 2 3 i d

Fig. 5. Growth curves of MCT-75 transfiirmed with plasmids pHCA- 159, pHCT-124 and pMCA-231. Plasmid pMCA-231 was cut with Hind11 1 before transformation. Cells were grown in yeast nitrogen base with 1 % methanol as carbon source for 4- 5 days and the cell density was determined spectrophotometrically. ( W ) Wild type; ( 0 ) pMCA-231; (0) pHCA-159; (0) pHCT-124

of catalase T transformants as indicated by the slight increase in absorbance at 600 nm (Fig. 5). However, in view of the fact that the cytoplasmic catalase T activity in transformed cells with methanol as carbon source is about 4 times higher than in yeast cells transformed with the catalase A gene (Table I), we conclude that localization of catalase within the peroxisomes as shown for catalase A is a prerequisite for peroxisomal function, i.e. for growth on methanol.

Expression of cutuluse A under control qf the methunol oxiduse promotcr

In order to obtain high-level expression of catalase A that could be regulated by glucose repression the catalase A gene

Fig. 6. Immunological detection ofcatalase A in peroxisomes of MCT- 75 transformed with plasmid pMCA-231 cut with HindIII. Cells were grown in yeast nitrogen base with 1 % methanol as carbon source and peroxisomes wcre isolated as described in Fig. 3. The four peak fractions exhibiting the highest methanol oxidase activities were sub- jected to polyacrylamide elecirophoresis in the presence of sodium dodecyl sulfate (50 pg protein each) and stained with Coomassie blue (A). Positions of the major peroxisomal proteins methanol oxidase (MOX) and dihydroxyacetone synthase (DAS) are marked. Immunoblot analysis of the gel (B) shows the (inactive) catalase of H. polymorphu (cHp) and the heterologous catalase A (cA), since the antibody is specific for catalases in both species. For molecular mass markers (kDa) see Fig. 3

was placed under control of the strong methanol oxidase promoter. Since multi-copy expression led to growth inhi- bition of transformed cells and poor reproduction of catalase activities, the vector pMCA-231 was cut with Hind111 before transformation in order to obtain single-copy integration that was previously shown to occur at random sites in the H . polymorphu genome 1151. Crude extracts of such transform- ants exhibited a constant catalase A activity of about 1400 U/ mg protein at derepressed conditions and 3200 U/mg protein under inducing conditions. Thus the specific enzyme activity was almost two times higher than wild-type and about six times higher than with the authentic promoter of the catalase A gene using a multi-copy plasmid (Table 1). The enhanced expression was also verified by immuno-blots showing a stronger signal of the catalase A protein band (Fig. 3, pMCA- 231).

In order to test whether the enhanced catalase A ex- pression in H . potyymorphu could improve the growth rates with methanol as a carbon source, growth of transformants was compared to that of wild-type cells. As shown in Fig. 5, no difference between the growth curves for wild-type and pMCA-231-transformed cells could be detected. Thus, a higher level of catalase A expression completely restores peroxisomal function. According to our notion stated above

178

that peroxisome function could not be complemented by the cytosolic catalase T activity, efficient import of the catalase A into the organelles was indicated by the improved growth on methanol. This could be demonstrated directly by isolation of peroxisomes on sucrose gradients and subsequent immuno- blot analysis of the catalase proteins. As depicted in Fig. 6, the catalase A band paralleled the peroxisomal marker proteins methanol oxidase and dihydroxyacetone synthase relative to staining intensities. Furthermore, the immuno-blot showed that the (inactive) H. polymorphu catalase that was always detected in crude extracts of transformed cells (Fig. 3) was still imported into the peroxisomes. If staining activities of the catalase A and the (inactive) H . yolymorpha catalase in crude extracts (Fig. 3) were compared with those of the peroxisome fractions (Fig. 6B), it can be concluded that the heterologous catalase A is imported as well as the authentic catalase.

DISCUSSION

Here we have described the isolation of a H . polymorpha mutant defective in peroxisomal catalase which is obligatory for the utilization of methanol as a carbon source. Further- more, we demonstrated that heterologous import of the catalase A from S. cerevisiue resulted in complementation of peroxisome function.

Since the mutant M-75 imports inactive catalase protein into the peroxisomes and can be functionally complemented by a heterologous catalase, it obviously carries a structural gene mutation. In contrast to S. cerevisiue that contains the peroxisomal catalase A and the cytoplasmic catalase T [I 0, 281, only one catalase is present in H . polymorphu. This was shown by the facts that no residual catalase activity is found in M-75 and that during enzyme purification only single activity peaks were observed (our unpublished data). Furthermore, only a single band of catalase in crude extracts could be detected immunologically.

The heterologous expression of catalase in H . polymorphn under control of their authentic promoters is constitutive, although both in S. cerevisiae [29] and in methylotrophic yeasts [17] catalases are subject to glucose repression. Thus, control mechanisms of gene regulation are probably different in both yeast species. Besides phylogenetic diversity this may be related to the fact that H . polymorphu is an aerobic yeast in contrast to S. cerevisiae. The participation of oxygen in catalase regulation for S. cerevisiue has been discussed [30].

An exceptional phenomenon was the high activity of catalase T in H. polynzorpha with methanol as carbon source. Since induction of the heterologous enzyme by methanol is extremely unlikely, it can be supposed that the high peroxide levels have an inducing effect on the enzyme synthesis. More detailed experiments including studies on transcription will be necessary to clarify this point.

Our results have clearly shown that import mechanisms in both yeast species are highly conserved despite the significant functional diversity of the peroxisome types. Import of catalase A into peroxisomes of H . polymorphu is very efficient, regardless of whether the gene is regulated by glucose ex- pression, as is the biosynthesis of peroxisomes [31], or constitutively expressed unlike peroxisome biosynthesis. Fur- thermore, import capacities of H . polymorpha peroxisomes are sufficient to translocate higher amounts of catalase than present in wild-type cells. However, as is to be expected, peroxisomal function depends on the amount of catalase, as

was shown by reduced growth rates on methanol if the catalase level is lower than in wild-type cells.

The compatibility of catalase import pathways in both yeast species may concern a variety of putative cellular components such as: (a) peroxisomal membrane receptors in analogy to those suggested [32] or shown for other membrdne translocation processes in eucaryotes [33 - 351; (b) specific targeting signals within peroxisomal proteins [36,37]; (c) cyto- plasmic factors maintaining import competence in eucaryotic and procaryotic systems [38 -441; (d) capacities of protein assembly into an active configuration as recently reported for yeast peroxisomes [45]; (e) heme synthesis and addition to the apo-enzyme [46]. The last two points could be most critical, since heterologous import of methanol oxidase into peroxisomes of S. cerevisiae was not correlated with enzymatic activity due to a lack of subunit assembly [47]. Possibly, the efficient complementation of peroxisome function as we have described here is based on a full compatibility of catalase A with the import machinery in H. polymorpha. This includes oligomerization of catalase A monomers that is required for catalytic activity, as has been demonstrated for bovine liver catalase [48].

Heterologous import of a peroxisomal protein was first demonstrated by immunofluorescence studies of firefly lu- ciferase expressed in mammalian cells [49]. The studies led to the discovery of a conserved targeting signal consisting of amino acids S, K and L at the carboxy terminus [36]. The same signal was also elucidated for peroxisomal proteins in rat liver [37] whereas certain yeast peroxisomal proteins contain targeting signals within two distinct protein domains [50]. Likewise, the two sequence of methanol oxidase and dihydroxyacetone synthase of H . polymorpha do not have an SKL sequence at the carboxy terminus [51, 521. However, the catalase A of S. cerevisiue shows an SKL sequence at the N- terminus [25]. Cloning and sequencing of the H . polymorpha catalase gene and comparative studies with the catalase A gene of S. cerevisiue will offer the possibility to verify SKL or another stretch of amino acids as a common targeting signal of these two yeast catalases.

We are most grateful to Drs A. Hartig and H . Ruis (University of Vienna, Austria) for providing the catalase A and T clones and for communicating sequence data prior to publication. We thank M. Eckart for providing plasmid pMEX and C.P. Hollenberg for his interest and criticism. This work was supported by the Deutsche FoPschurz~sgemeinschafi by grant Ro 66211-2 to R. R.

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