Peroxisome-deficient mutants of Hansenula polymorpha

YEAST VOL. 6: 87-97 (1 990)

Peroxisome-Deficient Mutants of Hansenula ~ o l y ~ o r ~ ~ a JAMES M . CREGG*, IDA J. VAN KLEIt, GRIETJE J. SULTERt, MARTEN VEENHUISS AND WIM HARDER?

The Salk Institute Biotechnologyllndustrial Associates, Inc., La Jolla, C A 92037, U.S.A. +Department ofMicrobio1og.v and $Laboratory for Electron Microscopy, Biological Center, University of Groningen, 97.51 N N Haren, The Netherlands

Received 14 June 1989; revised 16 October 1989

As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 960 methanol-utilization-defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase.

Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol- and ethanol- utilizing-deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome-deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol.

The mutations complemented and therefore were recessive mutations in different genes.

K ~ Y WORDS - Hansenula polymorpha; methylotrophic yeast; microbodies; peroxisome-deficient mutants; alcohol oxidase

INTRODUCTION Eukaryotic cells possess a variety of organelles, each devoted to performing a specific set of metabolic functions. The cell must maintain each of these organelles and assure that at least one of each is faithfully passed on to future generations. Further- more, each organelle requires a specific set of proteins which must be correctly addressed to the appropriate organelle. How the cell manages these operations for some organelles such as mitochondria, endoplasmic reticulum and vacuoles has been the subject of intensive investigations (Douglas rt ul., 1986; Kalderon et al., 1984; Schatz and Butow, 1983). Relative to these organelles, little is known about microbodies (peroxisomes, glyoxysomes; for reviews, see Fahimi and Sies, 1987; Veenhuis and Harder, 1988; Lazarow and Fujiki, 1985). Peroxisomes do not appear to arise from the endoplasmic reticulum or to be synthesized de now, *Present address: Department of Chemical and Biological Sciences, Oregon Graduate Center, Beaverton, OR 97006, U.S.A. :Addressee for correspondence.

0749-503X~90/020087-11 $05.50 0 1990 by John Wiley & Sons Ltd

but develop from pre-existing organelles (Veehuis et al., 1979). Proteins destined for peroxisomes are synthesized on free polysomes and post- translationally imported, without proteolytic pro- cessing (Borst, 1986, 1989). Recent results suggest that a tripeptide sequence, serine-lysinelhistidine- leucine, located at or near the carboxy-terminus of many peroxisomal matrix proteins, is both necessary and sufficient to direct proteins to the organelle (Gould et al., 1987, 1988, 1989).

The existence of yeast mutants that are defective in peroxisomal function would be of considerable value in research on peroxisome biogenesis and assembly. Among yeasts, the methylotroph Hanse- nula polymorpha is attractive for initiating such genetic studies for at least two .reasons. Firstly, the proliferation and physiological function of peroxisomes are easily controlled in this yeast by manipulating growth conditions (Veenhuis et al., 1983; Zwart, 1983; Veenhuis and Harder, 1987a). In particular, they are strongly induced during growth of cells on methanol (taking up to 80% of the total cytoplasmic volume); under these conditions the

88 J. M. CREGG ET AL.

organelles play an essential role in the metabolism of this compound since they mainly contain alcohol oxidase and dihydroxyacetone synthase (DHAS), key enzymes of methanol metabolism (Douma et al., 1985). Secondly, methods for both classical- and molecular-genetic manipulation have been described for H . polymorpha (Gleeson and Sudbery, 1988a,b; Roggenkamp et al., 1986; Tikhomirova et al., 1986; Cregg, 1987; Gleeson et al., 1986). Im- portantly, the isolation of genes by transformation and complementation of a mutant defect has been reported in this yeast species (Tikhomirova et al., 1988).

In this paper, we describe the isolation of two H . polymorpha mutant strains that appear to be completely devoid of peroxisomes. We report the screening procedure and the effect these mutations have on the activity and subcellular location of different enzymes normally present in the peroxisomal matrix.

MATERIALS AND METHODS

Microorganism and growth conditions

The wild-type Hansenula polymorpha strain was CBS4732. The following media were used: YPD = 1 YO yeast extract, 2% peptone, 2% glucose; CT =carbon source test medium, 0.67% yeast nitrogen base without amino acids, 0.1% yeast extract, 0.1 YO casamino acids, 50 pg/ml tryptophan; MM = mineral medium described before (Veenhuis e f al., 1979); CT and MM media were supplemented with a carbon source as designated in the text.

Mutant isolation

Procedures for mutagenesis and isolation of methanol-utilization-defective (Mut-) mutants were adapted from those described by Sanchez and Demain (1977) and Gleeson and Sudbery (1988b). Cultures of H. polymorpha were grown at 37°C overnight on a shaker in 250ml YPD, harvested from the exponential growth phase at a density of between 0.5 and 1 .O OD,,, units/ml, washed twice by centrifugation with 0.1 M-sodium citrate buffer (pH 5.5) and suspended in 50 ml of the same buffer with 100 pg/ml N-methyl-N'-nitro-N-nitrosoguani- dine (NTG). The cell suspension was held at room temperature without shaking and 25 ml aliquots were taken after 10 and 30 min of NTG treatment. NTG-treated cells were washed three times in sterile water and then inoculated into separate 70 ml YPD

media. After 2-6 h at 37"C, the cultures were harvested, concentrated by centrifugation and suspended in fresh YPD to approximately lOOD,,, units/ml. Glycerol was added to 30% final concen- tration and 2.0ml aliquots were dispensed into cryotubes, frozen and stored at - 70°C. The NTG treatment resulted in the death of approximately 99% of the cells and freezing killed an additional 90% of cells.

To screen for Mut- strains, mutagenized frozen cells were thawed, washed twice in 25 ml sterile water and spread on several CT medium agar plates supplemented with 0.1 YO glucose at a dilution that resulted in lO(r1000 colonies per plate. Plates were incubated at 37°C for 2 days and the colonies formed were replica-plated onto two sets of CT agar plates, the first supplemented with 0.5% methanol and the second with 0.5% glucose. Plates were incubated for 2 days at 37°C and colonies that failed to grow on methanol were picked and streaked onto YPD agar plates. Approximately 3% of the surviving mutagenized cells appeared to be Mut- after this initial screen. All potential Mut- strains were then patched onto CT agar with 0.1 YO glucose and screened a second time as before with special regard to tightness of phenotype, reversion frequency and growth rate on glucose. About 30% of strains selected during the first screen were judged satisfac- tory in the second screen and were kept for further analysis.

Carbon source utilization tests

Mut ~ strains were patched onto YPD agar and grown overnight at 37°C. To test for growth on ethanol, the strains on YPD were replica-plated onto CT medium supplemented with 0.1 YO glucose. After overnight incubation at 37"C, the strains were further replica-plated onto a series of CT agar plates supplemented with the following carbon sources: no carbon source, 0.5% ethanol, 0.5% glycerol and 0.5% glucose. After 2 days at 37"C, the strains were scored for growth.

Cell fractionation

Homogenized protoplasts of the two peroxisome- deficient strains and of diploids resulting from their mating were subjected to differential centrifugation as described by Douma et al. (1985). The 30,000 x g organellar pellet and the remaining supernatant were tested for sedimented and soluble peroxisomal proteins.

PEROXISOME-DEFICIENT MUTANTS OF HANSENULA POL YMORPHA 89

Enzyme assays Cell-free extracts were prepared as described by

de Koninget al. (1987). Alcohol oxidase activity was measured according to the method of Verduyn et al. (1984). Catalase activity was assayed as described by Luck (1 963), malate synthase and isocitrate lyase activities as described by Dixon and Kornberg ( 1959). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis was carried out as described by Laemmli (1 970). Western blotting experiments were performed using the protoblot immunoblotting system (Promega Biotec).

Electron microscopy Whole cells were fixed in 1.5% KMnO, for 20 min

at room temperature. Spheroplasts and subcellular fractions were fixed in 6% glutaraldehyde in 0.1 M- sodium cacodylate buffer, pH 7.2 for 60 min at O'C, followed by postfixation in a mixture of 0.5% OsO, and 2.5% K,Cr,O, in the cacodylate buffer for 90 min at 0°C. After dehydration in a graded ethanol series the samples were embedded in Epon 812; ultrathin sections were cut with a diamond knife and examined in a Philips EM 300.

C?>tochemistry and immunocytochemistry Cytochemical staining experiments for the sub-

cellular localization of catalase and alcohol oxidase on intact cells and spheroplasts were performed by the methods described previously (van Dijken et al., 1975; Veenhuis et al., 1976).

For immunocytochemistry, intact cells were fixed in 3% glutaraldehyde in 0.1 M-sodium cacodylate buffer, pH 7.2 for 90 min at O"C, dehydrated in a graded ethanol series and embedded in Lowicryl K4M (Zagers et al., 1986). Immunolabeling was performed on ultrathin sections with specific antibodies against alcohol oxidase (Douma et al., 1985) by the protein A/gold method described by Slot and Geuze (1984).

Other methods Genetical manipulation procedures such as

matings, complementation testing, sporulation and random spore analysis were performed as described in Gleeson and Sudbery (1988b).

RESULTS Isolation of methanol- and ethanol-utilization- defective mutants

Cultures of H. polymorpha were subjected to NTG mutagenesis and surviving cells were screened

for growth on methanol, as described in Materials and Methods. Approximately 1 YO of surviving cells were defective in methanol utilization (Mut-); 260 of these Mut- strains were collected and subjected to further analysis. In addition to the Mut- phenotype, it was anticipated that PER mutants may not be able to utilize ethanol as carbon source, the metabolism of which is known to involve the microbody matrix enzymes isocitrate lyase and malate synthase (Zwart, 1983; Zwart et a[., 1983; Veenhuis and Harder, 1987a). Each strain was therefore examined for growth on ethanol as sole carbon and energy source. Of the 260 Mut- strains, 62 were also defective in ethanol utilization (Eut-).

Identi$cation of peroxisome-deficient mutants

To identify putative peroxisome-deficient mutants, the overall cell morphology was examined by electron microscopy. This procedure would be too laborious to perform on the entire collection. Therefore, efforts were concentrated on those strains that were simultaneously Mut- and Eutr . These mutants were examined for the presence of peroxisomes after extensive precultivation in glucose medium (Veenhuis et al., 1979) followed by incubation for 24 h in methanol-containing media. In wild-type cells these conditions lead to the induc- tion of these organelles, which at the subcellular level are easily recognized by their large crystalline inclusions, consisting of the enzyme alcohol oxidase (Veenhuis et al., 1981; Veenhuis and Harder, 1987a). All mutants tested contained varying levels of alcohol oxidase activity, which was localized in peroxisomes (data not shown) with the exception of two strains, namely the mutant strains C27 and CI 11, which appeared to be peroxisome-deficient. The absence of recognizable peroxisomal structures in these strains was established by screening through several series of serial sections through glucose- grown and methanol-induced cells of both strains. A typical example of a glucose-grown C27 cell is shown in Figure 1. In wild-type (control) cells the small characteristic peroxisomal structures of glucose-grown cells (Figure 3) and those induced during incubations in methanol (Figure 3, inset) were readily detected. However, in identically prepared cultures of C27 and C111, recognizable peroxisomal profiles were never observed (Figures 2 and 4). Similar results were obtained with cells grown on glycerol or cells incubated in ethanol- containing media.


Figures 1 4 . Survey of cells of both mutant strains, grown on glucose (Figure I ; C27) or methanol (Figure 2; CI 11 and Figure 4; C27), showing the overall cell morphology and the absence of peroxisomal profiles. Typical peroxisomal profiles of wild-type cells, grown in batch cultures under the same conditions, are shown in Figure 3 (glucose; inset: methanol). Peroxisomal profiles are indicated by arrows.

Figures 1-6 and 8-1 1 are electron micrographs of intact cells of wild-type and peroxisome-deficient mutant strains of H. polymorpha, fixed with KMnO,, unless otherwise indicated. The marker represents I pm. Abbreviations: N = nucleus, M =mitochondrion, V = vacuole.

Also after cytochemical staining for catalase activity, a microbody marker enzyme in H . polymorpha, peroxisomal profiles were not detected in either of the mutant strains grown under various conditions despite an extensive search of serial sections (Figure 5). Since in the wild-type strain the small peroxisomes, present in glucose-grown (not shown; see Veenhuis et al., 1979) or ethanol-grown cells (Figure 6) and the large peroxisomes character-

istic for methanol-grown cells were readily detected after this procedure (Figure 6), we concluded that both mutant strains lacked recognizable peroxisomal structures.

Activities and subcellular localization of peroxisomal enzymes

The presence of different peroxisomal matrix enzymes was tested by both biochemical and


Figures 5 and 6. Cytochemical staining for catalase activity. Figure 5. Strain C111 incubated for 24 h on methanol, lacking recognizable peroxisomes. In controls, performed on a 1 : 1 mixture of methanol- and ethanol-grown wild-type cells, peroxisomes are readily detected in both the methanol-grown (Figure 6; cell at left hand side) and ethanol-grown (Figure 6; cell at right hand side) cells.

Table 1 . strains of H . polymorpha

Activities of different peroxisomal matrix enzymes in wild-type and mutant

Growth condition

Methanol Ethanol

Alcohol Isocitrate Malate Strain Catalase oxidase lyase synthase

Wild type 190.0 4.7 0.85 0.21 c27 28.5 0.003 0-02 0.22 C l l l 45.5 0.2 0.63 -

Diploid (C27 x C111) 143.9 12.3 0.61 0.90

Data presented are derived from cells incubated for 24 h in methanol or ethanol media. Catalase activity is expressed as AEz4 min-' per mg protein; alcohol oxidase, malate synthase and isocitrate lyase activities as U mg protein-'. - = activity not detectable.

immunochemical methods. In cell-free extracts of both mutant strains which had been incubated for 24 h in methanol- or ethanol-containing media, activities of catalase, malate synthase and isocitrate lyase were detected although at reduced levels com- pared to wild-type cells (Table 1). Apart from these enzymes, alcohol oxidase activity was found in strain C111 and in extremely low amounts in strain C27. The presence of these proteins, except for isocitrate lyase, for which no specific antibodies were available, was confirmed in Western blotting experiments (Figure 7A). These experiments further indicated that DHAS protein was present in both mutant strains (Figure 7A); however, whether this enzyme shows activity in these strains is not yet known. In addition, both strains contained formaldehyde- and formate dehydrogenase activities,

enzymes which are involved in further methanol dssimilation and which are known to be located in the cytosol (Douma et al., 1985). Apart from these enzymes, both strains also contained different peroxisomal membrane proteins, as was indicated by Western blotting (Figure 7B).

Fractionation studies were performed in order to determine the subcellular localization of the peroxisomal matrix enzymes. After differential centrifugation of homogenates of protoplasts of wild- type cells, these enzymes were sedimented and, as expected for peroxisome-bound enzymes (Douma et al., 1985), were present mainly in the 30,000 x g pellet (Table 2); however, in both mutant strains these enzymes were present in the supernatant, indicating a soluble nature and suggesting a cytosolic origin.


Figure 7. (A) Western blot of different peroxisomal matrix proteins in cells of wild-type and peroxisome- deficient strains C27 and CI 11 of H. polymorpha incubated for 24 h in media containing 0.5% methanol (for the detection of alcohol oxidase and DHAS protein) or 0.5% ethanol (for the detection of malate synthase protein). Equal amounts of protein were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with polyclonal antibodies against alcohol oxidase (75 kD), DHAS (76 kD) and malate synthase (62 kD). (B) Western blot of peroxisomal membrane proteins (24, 31, 51 and 62 kD respectively) from the same three strains tested after incubation with polyclonal antibodies prepared against the peroxisomal membrane of methanol-grown cells (for details on the polypeptide composition of the peroxisomal membranes of H . polymorpha, see Sulter et a/.. 1989).

Table 2. Distribution patterns of different peroxisomal enzymes after differential centrifugation of homogenized cells of wild-type and peroxisome-deficient strains of H. polymorpha. Cells were extensively precultivated on glucose, subsequently transferred into media containing 0.5% methanol or 0.5% ethanol as the sole carbon source and incubated for 24 h. Data are expressed as the ratio of the specific activities present in the 30,000 x g supernatant and 30,000 x g pellet fraction

Growth condition

Strain

Methanol Ethanol

Cyt. c Alcohol Cyt. c Isocitrate Malate oxidase oxidase Catalase oxidase Catalase lyase synthase

Wild type 0.1 1 0.10 0.3 1 0.06 0.37 0.5 1 0.34 5 2 C27 0.13 - 3.27 0.09 5.3

C l l l 0.18 13.5 8.09 0-09 3.26 2.22 - Diploid (C27 x C111) 0.06 0.76 1.16 0.06 4.1 4.36 4.82

-

Cells were harvested, converted to spheroplasts and their homogenates centrifuge at 2800 x g for I0 min, 9500 x g for 10 min and 30,000 x g for 20 min. Activities were expressed as U mg protein- I, except for catalase which is given as AE,,, min-' per mg protein (for specific activities present in crude extracts see Table 1). - =activity not detectable.

Further evidence for a cytosolic localization of alcohol oxidase was provided by (immuno)cytochemical results. After immunocytochemical experiments using specific antibodies against alcohol

oxidase, specific labeling was confined to the cytosol (Figure 8). The cytosolic nature of alcohol oxidase activity was further confirmed cytochemically; after incubations of protoplasts with CeCl, and methanol

PEROXISOME-DEFICIENT MUTANTS OF HA NSENULA POL YMORPHA 93

Figures 8 and 9. Cells of the mutant strain C111, showing (i) the cytosolic localization of alcohol oxidase indicated by the exclusive cytosolic distribution of gold particles after incubation of ultrathin sections of Lowicryl-embedded cells with anti-alcohol oxidase and protein A/gold (Figure 8) and (ii) the exclusive cytosolic localization of enzyme-specific reaction products after incubations for the detection of alcohol oxidase activity (Figure 9; CeC1, and methanol; glutaraldehyde-OsO,/K,Cr,O,). Staining of the mitochondria1 cristae is. as in diaminobenzidine-based experiments (van Dijken et al., 1975), due to cytochrome C peroxidase activity.

Figures 10 and 1 1. Ultrathin section through a diploid cell (C27 x C111) grown on ethanol, showing the presence of peroxisomes in the cell (Figure 10; arrow). In methanol-grown diploid cells the organelles are positively stained after incubations for the detection of alcohol oxidase activity (Figure 11; CeC1, and methanol; glutaraldehyde-OsO,/K,Cr,O,).

the alcohol oxidase reaction products were observed in the cytosol only (Figure 9).

Genetic analysis Strains C27 and CI 11 were mated and the result-

ing diploids were selected by their capacity to grow on either ethanol or methanol. During growth

under these conditions the diploids expressed high levels of activity of alcohol oxidase and other peroxisomal matrix enzymes (Table 1). Electron microscopical analysis revealed that in methanol-grown diploids, alcohol oxidase activity was localized in peroxisomes, the number and shape of which were similar to those in wild-type cells (Figures 10 and


11). Thus, the mutations that were responsible for peroxisome deficiency in C27 and C1 1 1 were recessive and in different genes.

To determine whether peroxisome deficiency was linked to either Mut- or Eut- or both phenotypes, C27 and C111 were each mated with adell and met6 auxotrophic strains of H . polymorpha (Gleeson and Sudbery, 1988b). After selection for diploids on CT medium with methanol as carbon source, the resulting diploids were sporulated and subjected to genetic analysis. Tetrad analyses were inconclusive because of the low viability of spores from these crosses. Therefore, spore products were analyzed by the random spore procedure (Gleeson and Sudbery, 1988b). We observed that for all crosses involving either C27 or C111, the Mut- and Eut- phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. Progeny from C1 1 1 crosses, five Mut- Eut' and five Mut' Eut- strains, were examined under the electron microscope for the presence of peroxisomes. No recognizable peroxiso- ma1 structures were detected in any Mut- Eut' strain, while all Mut' Eut- strains contained normal peroxisomes. An identical analysis of C27 spore products yielded the same general result. As expected, each Mut' Eut- spore product harboured normal peroxisomes. However, of the 12 Mut- Eut' spore products, eight displayed peroxisomal structures whereas the other four were peroxisome-deficient. To analyse this phenomenon, these 12 Mut- Eut' strains were subjected to complementation analysis. The results revealed that C27 contained complementing Mut mutations in two different genes, which we will refer to here as mutl and mut2. Analysis of cells incubated for 24 h in methanol-containing media revealed that those strains which harboured both mutations (four strains) were peroxisome-deficient; strains that were mutl alone (four strains) had virtually normal peroxisomes while mut2 alone (four strains) contained several very small peroxisomes, cytochemically characterized by the presence of catalase (not shown) together with cytosolic crystalloids of alcohol oxidase. These latter mutants will be described in detail in a separate paper.

Thus, peroxisome deficiency in C27 corres- ponded to the combination of mutl mut2 mutation. Taken together, we conclude that in C l l l the peroxisome-deficient and Mut - phenotypes were due to the effect of a mutation in a single gene, whereas in C27 mutations in two different genes caused the peroxisome deficiency.

DISCUSSION

This report describes an important step in a genetic approach towards understanding peroxisome biogenesis in yeasts. At least two questions relevant to the isolation of peroxisomal mutants (PER mutants) have been answered by this work. Firstly, in analogy to lethal human peroxisomal disorders (e.g. Zellweger syndrome: Goldfischer er al., 1973), it was a serious concern prior to this work that peroxisomes may be indispensable for cell viability under all growth conditions, and therefore, PER mutants could only be isolated as conditionally lethal strains. The isolation of PER mutants demonstrates that H . polymorpha strains without peroxisomes are viable. Secondly, although a number of metabolic pathways that involve one or more peroxisomal matrix enzymes are known in H . polymorpha (Veenhuis and Harder, 1987a), it was not clear prior to this study which, if any, strictly required peroxisomes.

This work demonstrates that screening for mutants unable to utilize methanol as sole carbon and energy source is a suitable strategy for the isolation of peroxisome-deficient mutants. In this respect the use of the methanol-defective phenotype to enrich for such mutants is in principle similar to the recently reported use of the oleate-defective phenotype to isolate the equivalent mutants in Saccharomyces cerevisiae (PAS mutants; Erdmann et al., 1989; Veenhuis et al., 1987b). Almost certainly other PER genes exist. Now that the phenotype of a PER mutant is clear, screening for other mutants will be more effective. An additional aid in mutant isolation would be a H . polymorpha strain capable of mating at high frequency. This would expedite mass complementation analysis of mutant collec- tions and thereby greatly reduce the number of strains to be examined by electron microscopy since only a single representative member of each complementation group needs to be tested. With the H . polymorpha CBS 4732 strain which was used in these experiments, mating frequencies were insuf- ficient for large-scale complementation analysis of mutants. Improved mating conditions were sought without success (data not shown). However, suffi- ciently high mating frequencies have been reported for H. polymorpha strain NCYC 495 (Gleeson and Sudbery, 1988b).

Since peroxisomal enzymes are active in the cytosol of peroxisome-deficient mutants (Erdmann et al., 1989; this work), why do such mutants not grow on certain substrates? The answer is not yet clear and may differ with each substrate. For


example, during methanol growth peroxisomes may be essential in the regulation of formaldehyde fluxes that are generated from methanol over dissimila- tory and assimilatory pathways (Douma et al., 1985). The known high affinity of formaldehyde for glutathione (Harder and Veenhuis, 1989) would render the formaldehyde inaccessible for DHAS, thereby preventing carbon assimilation from methanol and thus growth in PER mutants. In the case of oleic acid utilization-deficiency of PAS mutants of S . cerevisiae, peroxisomes may play a role in the formation and maintenance of carefully balanced multi-protein complexes necessary for the operation of the P-oxidation cycle.

In view of this, it is perhaps more surprising that different peroxisome-mediated pathways in fact operate quite well without peroxisomes. Firstly, we expected PER mutants to be defective in ethanol utilization-which they were not-since this path- way requires the microbody (glyoxysomal) enzymes isocitrate lyase and malate synthase (Zwart, 1983). Secondly, very recently we discovered that PER mutants are able to grow at wild-type rates on glucose in the presence of an organic nitrogen source, the metabolism of which is known to involve a peroxi- soma1 enzyme (e.g. primary amines or D-alanine; Veenhuis and Harder, 1987a). As such, these are the first examples of peroxisome-mediated pathways which are fully operative in the cytosol. These experiments will be detailed in a separate paper (Sulter rt al., 1989).

Another unexpected characteristic of the PER mutants was the presence of active cytosolic alcohol oxidase in both mutant strains. Based on earlier findings, we anticipated that cells without peroxisomes would also be defective in alcohol oxidase activity. This was indicated by the fact that after expression of the H . polymorpha alcohol oxidase gene in S. cerevisiae, binding of flavin adenine dinuc- leotide (FAD) and assembly ofthe protein into active octamers was not observed, although precursors of alcohol oxidase were imported into microbodies (Disteletal., 1987). However, when thesamealcohol oxidase-expressing S. cerevisiae cells were fused to non-alcohol oxidase-expressing cells of H . polymorpha, alcohol oxidase activity was observed (van der Klei et al., 1989). Taken together, these results suggested that specific factors present in peroxisomes of H . polymorpha are required for FAD binding, alcohol oxidase octamerization and activation or both. The finding that alcohol oxidase is active in the cytosol of the peroxisome-deficient mutants indi- cates that these factors, if they exist, can function

in the cytosol and suggests that the specific acidic environment of the peroxisomal matrix (Nicolay et al., 1987) is not a prerequisite for their proper functioning.

To date, peroxisome-deficiency has been described in only a few other organisms: baker’s yeast (Erdmann et al., 1989), hamster ovary cells (Zoeller and Raetz, 1986) and humans (Goldfisher et al., 1973; Fahimi and Sies, 1987). Cultured cells from patients with Zellweger syndrome appear to be completely devoid of these organelles. Cell fusion experiments indicated that a mutation in any one of at least five different genes will result in the disorder (Brul et al., 1988). The PER mutants of H . polymorpha share a number of similarities with the PAS mutants of S. cerevisiue and Zellweger cells. Firstly, all mutant alleles described to date are recessive to their respective wild-type alleles. Secondly, in all mutants peroxisomal enzymes accumulate and are active in the cytosol. Thirdly, peroxisomal membrane proteins are still present in all mutants. In Zellweger cells membrane proteins appear to be associated with membranous structures, termed peroxisomal ‘ghosts’ (Santos et al., 1988). It is expected that the analogy between Zellweger cells and yeast peroxisome-deficient mutants is more than superficial and that yeasts will serve as valid model systems to study and understand molecular events surrounding the human disease state.

ACKNOWLEDGEMENTS We thank Ineke Keizer-Gunnink, Klaas Sjollema and Jan Zagers for skillful assistance in different aspects of the electron microscopy and Dr. Martin Gleeson for guidance with genetic analysis methods. Grietje Sulter was supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (NWO). James Cregg was supported by NWO and SIBIA.

REFERENCES Borst, P. (1986). How proteins get into microbodies

(peroxisomes, glyoxysomes, glycosomes)? Biochim. Biophys. Acta 866, 179-203.

Borst, P. (1989). Peroxisome biogenesis revisited. Bio- chim. Biophys. Acta 1008, 1-13.

Brul, S., Wiemer, E. A. C., Westerveld, A., Strijland, A,, Wanders, R. J. A,, Schram, A. W., Heymans, H. S. A,, Schutgens, R. B. H., van den Bosch, H. and Tager, J. M. (1988). Kinetics of assembly of peroxisomes after


(Eds), The Yeasts, vol. 3, 2nd edn. Academic Press, London, New York, pp. 289-3 16.

Kalderon, D., Roberts, B. L., Richardson, W. D. and Smith, A. E. (1984). A short amino acid sequence able to specify nuclear location. Cell 39,499-509.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227,680-685.

Lazarow, P. B. and Fujiki, Y. (1985). Biogenesis of peroxisomes. Annu. Rev. Cell Biol. 1,489-530.

Luck, H. (1963). Catalase. In Bergmeyer, H. Y. (Ed.), Methods in Enzymatic Analysis. Academic Press, New York, pp. 885-894.

Nicolay, K., Veenhuis, M., Douma, A. C. and Harder, W. (1987). A 3'P NMR study of the internal pH of yeast peroxisomes. Arch. Microbiol. 147,3741.

Roggenkamp, R., Hanson, H., Eckart, M., Janowicz, Z. and Hollenberg, C. P. (1986). Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol. Gen. Genet. 202,302-308.

Sanchez, S. and Demain, A. L. (1977). Enrichment of auxotrophic mutants in Hansenulapolymorpha. Eur. J . Appl. Microbiol. 4,4549.

Santos, M. J., Imanaka, T., Shio, H., Small, G. M. and Lazarow, P. B. (1988). Peroxisomal membrane ghosts in Zellweger syndrome-aberant organelle assembly. Science 239,15361 539.

Schatz, G. and Butow, R. A. (1983). How are proteins imported into mitochondria? Cell 32,3 1 6 3 18.

Slot, J . W. and Geuze, H. J. (1984). Gold markers for single and double immunolabeling of ultrathin cryo- sections. In Polak, J. M. and Varndell, J. M. (Eds), Immunolabeling for Electron Microscopy. Elsevier Science Publishers, Amsterdam, pp. 129-142.

Sulter, G. J., Looyenga, L., Veenhuis, M. and Harder, W. (1989). Occurrence of peroxisomal membrane proteins in methylotrophic yeasts grown under different conditions. Yeast, in press.

Tikhomirova, L. P., Ikonomova, R. N. and Kuznetsova, E. N. (1986). Evidence for autonomous replication and stabilization of recombinant plasmids in the transfor- mants of yeast Hansenulapolymorpha. Curr. Genet. 10,

Tikhomirova, L. P., Ikonomova, R. N., Kuznetsova, E. N., Fodor, I. I., Bystrykh, L. V., Aminova, L. R. and Trotsenko, Y. A. (1988). Transformation of methylotrophic yeast Hansenula polymorpha: cloning and expression of genes. J. Basic Microbiol. 28, 343- 351.

van der Klei, I. J., Veenhuis, M., van der Ley, I. and Harder, W. (1989). Heterologous expression of alcohol oxidase in Saccharomyces cerevisiae: properties of the enzyme and implications for microbody development. FEMS Microbiol. Letters 57, 133-138.

van Dijken, J. P., Veenhuis, M., Vermeulen, C. A. and Harder, W. (1975). Cytochemical localization of

74 1-747.

fusion of complementary cell lines from patients with cerebro-hepato-renal (Zellweger) syndrome and related disorders. Biochem. Biophys. Res. Comm. 152,

Cregg, J. M. (1987). Genetics ofmethylotrophic yeasts. In Duine, J. A. and van Verseveld, H. W. (Eds), Proceed- ings of the Fifth International Symposium on Microbial Growth on C, Compounds. Martinus Nijhoff Publishers, Dordrecht, The Netherlands, pp. 158-167.

De Konig, U., Gleeson, M. A., Harder, W. and Dijk- huisen, L. (1987). Regulation of methanol metabolism in the yeast Hansenula polymorpha. Arch. Microbiol.

Distel, B., Veenhuis, M. and Tabak, H. F. (1987). Import of alcohol oxidase into peroxisomes of Saccharomyces cerevisiae. EMBO J.6,3111-3116.

Dixon, G. and Kornberg, H. L. (1959). Assay methods for key enzymes of the glyoxylate cycle. Biochem. J . 72, 3-9.

Douglas, M. G., McCammon, M. T. and Vassarotti, A. ( 1 986). Targeting proteins into mitochondria. Micro- biol. Rev. 50, 166-178.

Douma, A. C., Veenhuis, M., de Koning, W. and Harder, W. (1985). Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha. Arch. Microbiol. 143,237-243.

Erdmann, R., Veenhuis, M., Mertens, D. and Kunau, W. H. (1989). Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA

Fahimi, H. D. and Sies, H. (1987). Peroxisomes in Biology and Medicine. Springer-Verlag KG, Berlin.

Gleeson, M. A., Ortori, S. and Sudbery, P. E. (1986). Transformation of the methylotrophic yeast Hansenula polymorpha. J. Gen. Microbiol. 132,3459-3465.

Gleeson, M. A. and Sudbery, P. E. (1988a). The methylotrophic yeasts. Yeast 4, 1-15.

Gleeson, M. A. and Sudbery, P. E. (1988b). Genetic analysis in the methylotrophic yeast Hansenula polymorpha. Yeast 4,293-303.

Goldfischer, S., Moor, C. L., Johnson, A. B., Spiro, A. J., Valsamis, M. P., Wisniewski, H. K., Ritch, R. H., Norton, W. T., Rapin, I. and Garner, L. M. (1973). Peroxisomal and mitochondria1 defects in cerebro- hepatorenal syndrome. Science 182,62-64.

Gould, S. J., Keller, G.-A. and Subrammani, S. (1987). Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J. Cell Biol. 105,

Gould, S. J., Keller, G.-A. and Subramani, S. (1988). Identification of peroxisomal targeting signals located at the carboxy terminus of four peroxisomal proteins. J . Cell Biol. 107,897-905.

Gould, S . J., Keller, G.-A,, Hosken, N., Wilkinson, J. and Subramani, S. (1989). A conserved tripeptide sorts proteins to peroxisomes. J. Cell Biol. 108, 1657-1664.

Harder, W. and Veenhuis, M. (1989). Metabolism of one- carbon compounds. In Rose, A. H. and Harrison, J. s.

1083- 1089.

147,375-387.

86,5419-5423.

2923-2931.


catalase activity in methanol-grown Hansenula polymorpha. Arch. Microbiol. 105,261-267.

Veenhuis, M., van Dijken, J . P. and Harder, W. (1976). Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol- grown Hansenula polymorpha. Arch. Microbiol. 111,

Veenhuis, M., Keizer, I . and Harder, W. (1979). Charac- terization of peroxisomes in glucose-grown Hansenula polyrnorpha and their development after the transfer of cells into methanol-containing media. Arch. Microbiol

Veenhuis, M., Harder, W., van Dijken, J. P. and Mayer, F. (1981). Substructure of crystalline peroxisomes in methanol-grown Hansenula polymorpha: evidence for an in vivo crystal of alcohol oxidase. Molec. Cellular Biol. I ( lo), 949-957.

Veenhuis, M., van Dijken, J. P. and Harder, W. (1983). The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv. Microb. Phjsicrl. 24, 1-82.

Vcenhuis, M. and Harder, W. (1987a). In Fahimi, H. D. and Sies, H. (Eds), Peroxisomes in Biology and Medicine. Springer-Verlag KG, Berlin, pp. 436-458.

Veenhuis, M., Mateblowski, M., Kunau, W. H . and Harder, W. (1987b). Proliferation of microbodies in Saccl~uromyces cerevisiae. Yeast 3,77784.

123-1 35.

120,167-175.

Veenhuis, M. and Harder, W. (1988). Microbodies in yeasts: structure, function and biogenesis. Microbiol. Sciences 5,347-351.

Verduyn, C., van Dijken J.P. and Scheffers, W.A. (1984). Colorimetric alcohol assays with alcohol oxidase. J . Microbiol. Methods 2, 15-25.

Zagers, J. , Sjollema, K. and Veenhuis, M. (1986). Con- struction and use of an improved, inexpensive cabinet for low temperature embedding of biological tissue for electron microscopy. Lab. Practice 35, 1 1 4 - 1 15.

Zoeller, R. A. and Raetz, C. R. H. (1986). Isolation of animal cell mutants deficient in plasmalogen bio- synthesis and peroxisomal assembly. Proc. Natl. Acad. Sci. USA 83,5110-5 174.

Zwart, K. B., Veenhuis, M., Plat, G. and Harder, W. (1983). Characterization of glyoxysomes in yeasts and their transformation into peroxisomes in response to changes in environmental conditions. Arch. Microbiol.

Zwart, K. B. (1983). Metabolic SigniJicance of Micro- bodies in the Yeasts Candida utilis and Hansenula polymorpha. PhD Thesis, University of Groningen, The Netherlands.

136,28-38.

Documents

Peroxisome-deficient mutants of Hansenula polymorpha