132

Biochimica et Biophysica Acta, 1034 (1990) 132 141 Elsevier

BBAGEN 23282

A rapid selection for animal cell mutants with defective peroxisomes Olivier H. M o r a n d *, L e e - A n n H. A l l e n *, R a p h a e l A. Z o e l l e r + and Christian R.H. Raetz * Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, W1 (U.S.A.) (Received 28 August 1989)

Key words: Peroxisome deficient mutant; Plasmalogen synthesis; 9-(l'-Pyrene)nonanol

Chinese hamster ovary (CHO) cells take up and incorporate 9-(l'-pyrene)nonanol (P9OH) into phospholipids and neutral lipids. Exposure of P9OH-iabeled cells to long wavelength ultraviolet (UV) light causes cell death, because excitation of the pyrene moiety generates reactive oxygen species. CHO mutant cells deficient in plasmalogen biosynthesis and peroxisome assembly (Zoeller, R.A. and Raetz, C.R.H. (1986) Proc. Natl. Acad. Sci. USA 83, 5170-5174) are much more resistant to P 9 O H / U V treatment than are wild-type cells. This phenotype is explained by a 7.5-fold reduction of P9OH incorporation into the ethanolamine-linked phospholipids in the mutant cells and 2.4- to 6-fold reduction of P9OH incorporation into all other phospholipids and triglycerides, suggesting a general defect in fatty alcohol metabolism. [U-14C]Hexadecanol incorporation into the phospholipids of the mutant cells is also impaired. In contrast, the fatty acid analog, 9-(l'-pyrene)nonanoic acid, is incorporated into cells two times more rapidly by the mutants than by the wild type. Resistance to P 9 O H / U V treatment affords a simple, new method for the selection of animal cell mutants defective in peroxisome biogenesis.

Introduction Peroxisomes were discovered more than two decades ago by de Duve and Baudhuin [1], who defined them as organelles containing catalase and at least one hydrogen peroxide-forming oxidase. Subsequent studies have shown that peroxisomes contain many other enzymes, depending on the cell type and animal species, including (i) all the enzymes required for the fl-oxidation of very long chain fatty acids. (ii) various enzymes of amino acid metabolism, and (iii) the enzymes catalyzing the initial steps of ether lipid biosynthesis [2,3].

Present addresses: * Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, U.S.A. + Biophysics Institute, Boston University School of Medicine, Boston, MA 02118, U.S.A. Abbreviations: P9OH, 9-(l'-pyrene)nonanol; P9, 9-(l'-pyrene)nonanoic acid; CHO cells, Chinese hamster ovary cells; PBS, phosphatebuffered saline; DHAP, dihydroxyacetonephosphate; UV, ultraviolet; NEM, N-ethylmaleirnide. Correspondence: C.R.H. Raetz, Merck Sharp and Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, U.S.A.

Many of the functions of peroxisomes in cell physiology and the mechanisms that govern their proliferation remain poorly understood [4,5]. Evidence for the diverse function of peroxisomes is provided by a group of human inherited diseases in which multiple peroxisomal enzyme activities are impaired [3] and the organelle is not properly assembled. These include Zellweger cerebro-hepato-renal syndrome, infantile Refsum's disease, neonatal adrenoleukodystrophy and hyperpipecolic acidemia, which present with severe, often lethal, abnormalities including neurological impairments and malformations. Although fibroblasts obtained from these patients have proven to be extremely useful for biochemical studies, the isolation of peroxisome-deficient mutants from continuously growing somatic cell lines seemed highly desirable for investigating the biogenesis of this organelle in more detail. Using colony autoradiography as a screening assay, Zoeller and Raetz [6] were able to isolate a set of Chinese hamster ovary (CHO) cell mutants defective in the peroxisomal enzyme, dihydroxyacetonephosphate acyltransferase. These mutants were further investigated and found to be deficient in peroxisomes based on assays of multiple enzyme activities, immunoblots of these enzymes, analysis of plas-

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133 malogen content and electron microscopy [6,39]. Among other things, peroxisomal DHAP acyltransferase, alkyl DHAP synthase, acyl-CoA oxidase and particulate catalase activities were very much reduced in these mutants, and their plasmalogen content was reduced 10-fold. Preliminary experiments from our laboratory showed that the incorporation of radiolabeled hexadecanol into peroxisome-deficient CHO cells was reduced when compared to wild-type cells treated under similar conditions [7]. This was explained, in part, by the inability of the mutant cells to synthesize plasmalogens. We postulated that the physiological fatty alcohols commonly used by normal animal cells could be replaced by fluorescent fatty alcohol analogs, such as 9-(l'-pyrene)nonanol (P90H), a photosensitizer [8]. We also hypothesized that normal cells, having the ability to incorporate the fluorescent fatty alcohol into lipids, would be killed when exposed to long wavelength UV light [8,9], but that mutants would incorporate P9OH poorly, rendering them less susceptible to photosensitized killing. This phenotype would then permit the selection of plasmalogen- and/or peroxisome-defective mutants from any cell line of interest. The present communication shows that wild-type CHO cells are indeed more sensitive to P9OH/UV killing than peroxisome-defective mutants. However, the absence of plasmalogens in these mutants is not the only reason for their reduced ability to incorporate P9OH.

Experimental procedures Materials. 9-(l'-Pyrene)nonanol, 9-(l'-pyrene)nonanoic acid and cholesteryl 10-(l'-pyrene)decanoate were purchased from Molecular Probes Inc. (Eugene, OR). [U-14C]Palmitic acid (31 GBq/mmol) and [4-14C]cholesterol (1.85 GBq/mmol) were purchased from Dupont-New England Nuclear. [2-14C]Ethanolamine (2.15 GBq/mmol) and [),-32p]ATP were from Amersham Corp., and [2-14C]acetate (sodium salt, 1.85 GBq/mmol) was from ARC Inc. (St. Louis, MO). [U14C]Hexadecanol was synthesized from [u-laC]palmitic acid, as described previously [10]. Dihydroxyacetone [32p]phosphate was synthesized according to Schlossman and Bell [11]. Bio-Safe II liquid scintillation fluid was obtained from RPI Corp. (Mount Prospect, IL). Silica gel 60 thin-layer chromatography plates (E. Merck) were purchased from American Scientific Products (McGraw Park, IL). Titanyl sulfate was obtained from Chemtech (Hayward, CA). All other reagents were purchased from Sigma (St. Louis, MO). Tissue culture flasks (Corning) and dishes (Falcon) were obtained from VWR Scientific (Bellwood, IL). Cells and culture conditions. All cell lines were grown at 37°C, in a 5% CO2/95% air atmosphere in Ham's F12 medium containing 10% fetal bovine serum (Gibco)

supplemented with 1 mM glutamine, penicillin G (100 units/ml), streptomycin (73.5 units/ml) and insulin (0.5 IU/ml). Wild-type Chinese hamster ovary cells (CHOK1) were obtained from the American Type Culture Collection. Strain ZR-82 was isolated as a plasmalogen/peroxisome-deficient mutant of CHO-K1 [6]. CHO-K1 cells were treated with ethylmethane sulfonate as previously described [12] and were cultured for several generations at 33 ° C, stored frozen in liquid nitrogen, and tested for the frequency of ouabain-resistant colonies prior to use in the selection procedure. When P9OH or P9 were added to cell cultures, a stock solution of the fluorescent probe was first prepared at a concentration of 20 mM in dimethylsulfoxide and was subsequently mixed into the growth medium to reach the appropriate concentration. The final concentration of dimethylsulfoxide to which cells were exposed did not exceed 0.1%, and its presence did not affect cell viability. P 9 O H / U V killing. Cells were seeded in 96-well microtiter dishes (Falcon) in 0.1 ml of growth medium at a density of 2.103 cells per well. 2 h after plating, 0.1 ml of growth medium containing increasing concentrations of P9OH was added to the wells, and the cells were incubated for 20 h at 37 o C. Then the cells were washed twice with 0.2 ml of growth medium, received 0.2 ml of fresh growth medium lacking P9OH, and were reincubated for 6 h. Prior to irradiation, the cells were washed once more and were again incubated in 0.2 ml of fresh growth medium. Cells were immediately irradiated by illumination for 2 min with two long wavelength ultraviolet fluorescent lamps (15W, Sylvania F1ST8 Blacklight Blue bulb) placed underneath at a distance of 2 cm. A 3 mm glass plate was positioned between the tissue culture dish and the UV source to eliminate ultraviolet light with wavelengths below 300 nm. The intensity at 365 nm was 3000 #W/cm 2 when measured through the plastic dish with a 365 nm BlakRay Ultraviolet meter (Ultraviolet Products Inc.). The plates were maintained for another 4-5 days in the incubator prior to quantitation of surviving cells using Crystal violet staining as described previously [13,14]. Alternatively, cells were seeded at low density (300 cells/60 mm diameter plate), allowed to attach for 24 h, and treated with P9OH and UV irradiation as described above. Colonies resulting from surviving cells were counted after staining with Coomassie blue, following outgrowth of the cells for 7-10 days. Selection of plasmalogen/peroxisome-deficient mutants. Mutagen-treated cells, originally grown at 33°C, were plated out in four 100 mm diameter tissue culture dishes (1 • 105 cells per dish). After 1 day at the permissive temperature (33°C), the growth medium was replaced by 15 ml growth medium containing 20 /~M P9OH. The cells were incubated for 20 h at 37°C, washed twice with fresh growth medium lacking P9OH

134 and reincubated for 6 h. Cells were washed once more and exposed to long wavelength ultraviolet light (365 nm, 1200-1600 /~W/cm 2) for 5 min. Surviving cells were allowed to grow for 12 days at 33°C, whereas dead cells and debris were eliminated by repeated medium changes no less than 24 h after irradiation. Surviving cells were harvested, replated and exposed once more to the P 9 O H / U V enrichment protocol. Resistant cells (P9OH R) were grown out, harvested, pooled and frozen away for storage in liquid nitrogen. P9OH R cells were thawed and seeded in tissue culture flasks prior to replating for colony growth and polyester overlay. Peroxisomal D H A P acyltransferase activity was assayed by colony autoradiography [6,15]. Assay. D H A P acyltransferase activity was determined in sonically irradiated cell pellets [6] using a modification of the methods of Schlossman and Bell [11]. Assays of catalase were performed according to the method of Peters et al. [16], as modified by Zoeller and Raetz [6]. The occurrence of plasmenylethanolamine in cells was assessed upon in vivo labeling with radiolabeled ethanolamine [7]. Briefly, cells were seeded in 60 mm diameter tissue culture dishes (1 • 105 cells/dish) in the presence of fresh growth medium containing [214C]ethanolamine (0.2 ~ C i / m l ) and incubated at 37 ° C. After 3 days, the cells were washed with phosphatebuffered saline (PBS), harvested with the aid of a rubber policeman and extracted in a single phase neutral Bligh and Dyer [17] solvent system [7]. Samples were converted into the two-phase partitioning system by adding PBS and chloroform. The lower (organic) phase was collected, evaporated to dryness under nitrogen, and analyzed by two-stage thin-layer chromatography in order to separate phosphatidylethanolamine and plasmenylethanolamine [7]. The radioactive phospholipids were localized by autoradiography, scraped off into scintillation vials containing 0.5 ml of methanol, and counted in 10 ml of Bio-Safe II by liquid scintillation spectrometry. Proteins were assayed according to Smith et al. [18]. Flow cytometry. Wild-type and mutant cells (2.5 • 106) were seeded in 15 ml of growth medium in 100 mm diameter plastic tissue culture dishes and placed in the 3 7 ° C incubator for 10-20 h. Next, the medium was removed, and cells received 10 /~M P9OH or P9 in 15 ml of fresh growth medium, and they were further incubated for 18 h. In one set of dishes, the medium was removed, and the cells were washed once with medium lacking fluorescent probe. Cells were harvested by trypsin treatment, centrifuged and washed three times by centrifugation in PBS. Cells resuspended in PBS were mixed with an equal volume of PBS 2% paraformaldehyde. After 30-45 min, cells were collected by centrifugation and washed once in PBS 0.02% sodium azide (w/v). Fixed cells were stored in PBS 0.02% sodium azide at 4 ° C prior to flow cytometry

analysis. In the second set of dishes, medium was removed following the incubation with P9OH or P9, and cells were washed once with medium and reincubated for 6 h in 15 ml of medium lacking fluorescent probe. Subsequently, cells were harvested and analyzed as described above. P9OH and P9 accumulations were analyzed using an EPICS-752 flow cytometer (Coulter, Hialeah, FL). The pyrene probe was excited by a single 363 nm UV line at 100 mW power. An LP-390 dichroic filter was used to separate scattered laser light from the pyrene fluorescence. A BP-488 dichroic filter was used to reflect the pyrene fluorescence to the photomultiplier. PMT voltage was set at 1,100 V. Fixed cells were passed at a rate of 500-1000 per s through a 76 /~m nozzle, using double-distilled water as the sheath fluid. A two-parameter histogram based on the forward angle and 90 ° laser light scattering was generated in order to differentiate intact cells from contaminating debris and clusters. Only fluorescent signals from intact cells were analyzed in the pyrene fluorescence histogram.

Incorporation of P9, P90H and [2-14C]acetate into cells. Wild-type and mutant cells (2.5 106) were seeded •

in 15 ml of growth medium in 100 mm diameter plastic tissue culture dishes and placed in the 37 ° C incubator for 10-20 h. Next, the medium was removed, and cells received 10 ~M P9 or P9OH in 15 ml of fresh growth medium, and they were further incubated for 18 h. In one set of dishes, the medium was removed, and the cells were washed once with 10 ml medium lacking fluorescent probe. Next, the cells were harvested with trypsin, washed twice by centrifugation in PBS and resuspended in 2 ml of PBS. A small portion of the cell suspension was used for protein determination [18], and 1.8 ml of the same suspension was mixed with 4 ml of methanol and 2 ml of chloroform [17], and heated at 6 0 ° C for 10 rain to facilitate lipid extraction. The single-phase extract was centrifuged, and the supernatant was collected and mixed with 2 ml of chloroform and 1.8 ml of water to achieve the proportions of the two-phase solvent system. The lower phase was collected, evaporated to dryness under nitrogen and analyzed by thin-layer chromatography in two different solvent systems: c h l o r o f o r m / m e t h a n o l / a c e t i c a c i d / water (25 : 15 : 4: 2, v / v ) for phospholipids, and nh e x a n e / d i e t h y l e t h e r / a c e t i c acid (80 : 2 0 : 1 or 6 0 : 4 0 : 1 , v / v ) for neutral lipids. In the second set of dishes, medium was removed following the incubation with P9OH or P9, and cells were washed once with medium and reincubated for 6 h in 15 ml of medium lacking fluorescent probe. Subsequently, cells were harvested and analyzed as described above. For photography, each silica gel plate was placed on top of a long wavelength UV light and covered with a 5 mm thick plexiglass plate to shield upcoming UV light. Black and white photographs were made with Kodak

135 Plus-X films using a Pentax Y2 yellow filter and a Kodak Wratten 2B-UV gelatine filter. In an attempt to better evaluate the differential incorporation of P9OH into cellular lipids, cells were also labeled simultaneously with P9OH and [2-14C]acetate in some experiments. Wild-type and mutant cells (1.105 ) were seeded in 5 ml of growth medium in 25 cm 2 tissue culture flasks and placed in the 37 ° C incubator for 4 h to achieve proper CO 2 equilibration. Then, 0.5 ml of medium containing 125 /~Ci/ml [2-14C]acetate (1.85 G B q / m m o l ) was added quickly to each flask to attain a final activity of approx. 11 /xCi/ml. Flasks were kept tightly closed thereafter and placed in the incubator at 37 °C. After 3 days of incubation in the presence of radiolabeled acetate, 0.5 ml of medium containing 120 /~M P9OH was added to obtain a final concentration of 10/~M, and cells were further incubated for 17 h. Next, the cells were washed once with medium, and they were reincubated in 5 ml of CO 2 pre-equilibrated medium, lacking P9OH and supplemented with 0.5 ml of radiolabeled acetate (final activity approx. 11/~Ci/ml). After 6 more hours of incubation at 37 ° C, cells were washed once with medium, harvested with 0.25% ( w / v ) trypsin in PBS, and centrifuged. The pellet was washed twice more by centrifugation in PBS, and the lipids were extracted and chromatographed as described above. The radioactive lipids were identified by autoradiography, and the fluorescent lipids were localized with the aid of a long wavelength UV light. All the radioactive a n d / o r fluorescent lipids were scraped off and extracted twice with 2 ml of c h l o r o f o r m / m e t h a n o l / w a t e r ( 1 : 2 : 0 . 9 , v/v). Next, each extract (3.9 ml) received 1 ml chloroform and 0.9 ml water to attain the proportions of a two-phase Bligh and Dyer system [17], followed by vortexing and centrifugation. The lower phases were collected, evaporated to dryness under nitrogen and redissolved in 0.5 ml of chloroform:methanol (95:5, v / v ) prior to measuring fluorescence [19]. Following fluorescence determination, each lipid extract was transferred into a scintillation vial, together with the wash from the original tube and the quartz cuvette. After evaporation, the extract was solubilized in 0.5 ml methanol and counted in 10 ml of Bio-Safe II by liquid scintillation spectrometry. Incorporation of [U- 14C]hexadecanol into cells. The incorporation of radiolabeled hexadecanol into cells was performed essentially as described previously [7]. Cells (1 • 105) were seeded in 1 ml of growth medium in sterile glass tubes and placed in the incubator for 6 h. An appropriate amount of [U-a4C]hexadecanol in toluene was mixed with nonradioactive hexadecanol to attain a final specific activity of 200 mCi/mmol, evaporated to dryness under nitrogen, dissolved in ethanol and finally mixed into growth medium. The medium in the tissue culture tubes was removed by aspiration, and the cells received 1 ml of the radioactive

solution. The final concentration of [U-14C]hexadecanol was 10 # M (2 /zCi/ml), and the ethanol concentration was 1% (v/v). In one set of tubes, the medium was removed by aspiration after 18 h of incubation, and the cells were washed twice by centrifugation in PBS. Finally, they received 0.9 ml of PBS. Each tube was then mixed with 2 ml of methanol and 1 ml of chloroform, and heated at 60 ° C for 10 min. Next, 1 ml of chloroform and 0.9 ml of water were added to obtain a two-phase Bligh and Dyer system [17]. The lower phase was collected and evaporated to dryness under nitrogen, and lipids were analyzed by thin-layer chromatography in the two different solvent systems: c h l o r o f o r m / methanol/acetic a c i d / w a t e r (25 : 15 : 4: 2, v / v ) and nhexane/diethyl ether/acetic acid (80: 20: 1, v/v). The radioactive lipids were localized by autoradiography, scraped off into scintillation vials containing 0.5 ml of methanol, and counted in 10 ml of Bio-Safe II by liquid scintillation spectrometry. Tubes containing untreated cells were used in parallel for protein determinations [20]. In another set of tubes, the medium was removed by aspiration after 18 h of incubation, and the cells were washed once with medium and then reincubated for 6 h in 1 ml medium in the absence of added hexadecanol. Lipid samples from these cells were analyzed as described above.

Identification of cholesteryl-9-(1 '-pyrene)nonanoate in P90H labeled cells. The putative fluorescent cholesterol ester (band 7) was extracted from the silica gel with c h l o r o f o r m / m e t h a n o l / w a t e r ( 1 : 2 : 0 . 9 , v / v ) and recovered by Bligh-Dyer partitioning, as described above. The sample was stored in chloroform at - 7 0 ° C prior to alkaline or acidic hydrolysis. For alkaline hydrolysis, a portion of the solution of band 7 was evaporated to dryness under N2, mixed with 0.2 ml of 1 M K O H in w a t e r / e t h a n o l (1:8, v / v ) and incubated at 7 0 ° C for 2 h. The extract was neutralized with 0.2 ml of 1 M HC1 and received 2 ml of chloroform, 1.8 ml of methanol and 1.6 ml of PBS. After vortexing and centrifugation, the lower phase was collected, evaporated to dryness and analyzed by thin-layer chromatography using nhexane/diethyl ether/acetic acid (80:20: 1, v/v). For acid hydrolysis, band 7 was evaporated to dryness, dissolved in 1 ml of chloroform, 2 ml of methanol and 0.8 ml of 25 mM HgC12 in 0.05 M HCI, and incubated at room temperature for 30 min. Next, the reaction was mixed with 1 ml of chloroform and 1 ml of PBS. After vortexing and centrifugation, the lower phase was collected, evaporated to dryness and analyzed by thin-layer chromatography using n-hexane/diethyl ether/acetic acid (80 : 20 : 1, v/v). In a separate set of experiments, cells (2- 105) were seeded in 0.5 ml of growth medium in sterile glass tubes and placed in the incubator for approx. 6 h. Next, each tube received 0.3 ml of radioactive cholesterol medium (prepared by mixing [4-14C]cholesterol (50 m C i / m m o l )

136 in ethanol with growth medium to obtain a concentration of 20 /~M cholesterol) and 0.2 ml of medium containing 50 # M P9OH, so that the final concentrations were 6 /~M [4-14C]cholesterol ( 0 . 3 6 / t C i / m l ) and 10 /~M P9OH. After 18 h at 37°C, the cells were washed once with growth medium lacking P9OH and twice with PBS. The lipids were extracted as described above. Samples were analyzed by thin-layer chromatography using n - h e x a n e / d i e t h y l e t h e r / a c e t i c acid (80 : 20 : 1, v / v ) . The radioactive lipids were localized by autoradiography, and the fluorescent bands were visualized by exposing the plate to long wavelength UV light. Results

Photosensitized killing of CHO cells using P 9 0 H Cells labeled with a pyrene-containing fatty acid are killed by long wavelength ultraviolet fight [21-23]. Excitation of the pyrene, a photosensitizer, at the appropriate wavelength is responsible for the formation of radicals and singlet oxygen from ground-state oxygen according to the so-called Type I and Type II chemistry [8,24]. Subsequently; reactive species of oxygen can rapidly damage proteins, lipids and D N A [8,9], resulting in cell death. The viability of CHO-K1 cells labeled with P9OH was greatly reduced by long wavelength UV irradiation (Fig. 1), and the extent of killing was dependent upon the concentration of P9OH employed to label the cells. A C H O mutant cell line defective in plasmalogen synthesis and peroxisome biogenesis [6]

was much more resistant to P 9 O H / U V treatment than were wild-type cells (Fig. 1). After 20 h of incubation with 8 ffM P9OH, followed by washing and 6 h of reincubation without P9OH prior to UV exposure, the viability of mutant ZR-82 was 70% of the control value (i.e., unlabeled cells exposed to UV), as opposed to 1% for the wild-type cells. All three of the independently isolated plasmalogen/peroxisome-deficient mutants [6] were resistant to P9OH treatment (data not shown). Labeling of the cells with P9OH up to 20 ~M had no toxic effect per se, as long as the cells were not exposed to long wavelength UV irradiation (data not shown). The 6 h reincubation in P9OH-deficient medium was required for selective killing of the wild-type cells. Without this step, the viability of both wild-type and mutant cells was less than 1% at 10/xM P9OH (data not shown).

P90H concentration (~M)

Isolation of plasmalogen / peroxisome-deficient mutants The selective resistance of the mutant cells to P 9 O H / U V killing indicated that this phenotype could be employed to isolate new mutants deficient in plasmalogens a n d / o r peroxisomes. Mutagen-treated C H O cells were seeded into 100 m m diameter tissue culture dishes at a density of 1 • 105 cells, and the selection of P 9 O H / U V resistant cells (P9OH R) was performed as described in Experimental Procedures. The first round of selection yielded 14-18 colonies per 105 mutagentreated cells, but only 1 - 2 colonies per 105 parental cells. The resistant, mutagen-treated cells were grown out at 3 3 ° C and subjected to a second round of P 9 O H / U V selection. At this point resistant cells were very numerous, making it impossible to count colonies. The survivors were then seeded at a low density in two 100 m m diameter dishes, overlaid with polyester cloth and glass beads, and cultured for 15 days at 3 3 ° C [6]. The colonies grown into polyester cloths were assayed for peroxisomal D H A P acyltransferase activity using colony autoradiography [6]. Of 184 colonies assayed, none was D H A P acyltransferase positive (data not shown). Six colonies were retrieved from the master dishes with the aid of cloning rings, grown at 33 ° C, and purified upon dilution into 96-well plates. Four of these lines, OM-522, OM-523, OM-524 and OM-612, were examined further.

Fig. 1. Sensitivity of P9OH labeled wild-type and mutant ZR-82 cells to long wavelength UV irradiation. Cells (2.103/well in 96-well microtiter dishes) in 0.2 ml of growth medium were incubated at 37°C for 20 h with the indicated concentrations of P9OH. The medium was aspirated and the cells were washed twice with fresh medium lacking P9OH and reincubated for 6 h in fresh medium lacking P9OH. The cells were washed once more and exposed to long wavelength UV for 2 min in 0.2 ml of medium as described under Experimental Procedures. The cells were allowed to grow for another 4 days, washed with PBS and stained for quantitation [13,14]. The survival rate is the percentage of the absorbance measured in each well relative to control UV-exposed unlabeled cells, and each point is the averagevalue of four determinations.

Characterization of the cloned P 9 0 H n mutants In normal animal cells, catalase is found primarily in the matrix of peroxisomes [25]. In Zellweger cells [26], as in peroxisome-defective C H O cells [6], catalase activity in whole cell homogenates is normal, but it is localized in the cytoplasm. When catalase activity was determined in the particulate and cytosolic fractions obtained from wild-type and P9OH R mutants (Table I), most of the catalase activity was detected in the particulate fraction prepared from wild-type cells, but in

lOO

c

8

80 6o

•~ 4o >

•~ 2o

4

8

12

16

20

137 TABLE I

T A B L E III

Catalase activities in wild-type and cloned P 9 0 H resistant mutant cells

Incorporation of [2-14C]ethanolamine into phospholipids of wild-type and cloned P 9 0 H resistant mutant cells

Catalase assays contained 10 m M imidazole-HC1 (pH 7.2), 0.5 m g / m l bovine serum albumin, 0.1% Triton X-100, 8.82 m M H202 and 20/~g protein in a total volume of 300 /~1. Reactions were performed at 25 ° C for 1 min and stopped by the addition of 3.0 ml of a titanyl sulfate solution. 1 unit of catalase activity is defined as the amount of enzyme required to degrade 90% of the H202 in 1 min. Each value is the average+ S.D. of five determinations. The total protein in the cytosolic and particulate fractions is about the same. Cells CHO-K1 OM-522 OM-523 OM-524 OM-612

Cytosolic catalase (units. mg p r o t - 1) 4.9 + 0.6 19.1 + 21.6 + 17.9 + 21.3 +

Particulate catalase (units. m g p r o t - 1) 19.2 + 2.0

2.4 2.1 2.3 1.9

2.2 + 0.6 2.4 + 0.7 2.6 +0.6 2.9 + 0.4

T A B L E II

D H A P acyltransferase activities in wild.type and cloned P 9 0 H resistant mutant cells Whole cell lysates were assayed at 30 o C in the presence or absence of N-ethylmaleimide (NEM) for 10-15 min. Each mixture consisted of 100 m M M e s , 100 m M N-tris(hydroxymethyl)methyl-2aminoethanesulfonic acid (pH 5.5 or 7.4, respectively), 100/~M palmitoyl-CoA, 1.5 m M [32p]DHAP ( 2 - 4 # C i / # m o l ) , 8 m M NaF, 5 m M MgCIE, 50 m M KC1, 2 m M KCN, bovine serum albumin at 2 m g / m l and 30-100 #g cell protein in a total volume of 300 #1. Each value is the average + S.D. of five determinations.

CHO-K1 OM-522 OM-523 OM-524 OM-612

(pmol. min - 1. mg prot - 1) pH 5.5

p H 7.4

( - )NEM

( - )NEM

( + )NEM

263 + 26

350 + 22

116 + 17

4+ 7+ 9+ 11+

2 3 5 3

325+33 578+36 382+44 375+35

Cells

Plasmenylethanolamine

Phosphatidylethanolamine

CHO-K1

35 690 + 4300

35 810 + 5 480

OM-522 OM-523 OM-524 OM-612

the P9OH R mutants, most of the catalase was found in the cytosolic fraction (Table I). As shown in Table II, the peroxisomal DHAP acyltransferase activity (measured at pH 5.5) in the mutants was less than 5% of the activity found in wild-type cells, consistent with the results of the colony autoradiography (see above). In contrast, the specific activity of the microsomal DHAP acyltransferase (measured at pH 7.4) [27] was very similar in wild-type cells and the P9OH R mutants (Table II). In the presence of NEM (a potent inhibitor of the microsomal isozyme), significant residual DHAP acyltransferase activity was detected in wild-type cells at pH 7.4, but not in the cloned P9OH R mutants (Table II), consistent with the absence of the NEM-insensitive peroxisomal isozyme. These observations are the same as those for the previously described mutants of Zoeller and Raetz [6].

Cells

Cells grown in 60 man diameter tissue culture dishes were incubated for 3 days in the presence of [2)4C]ethanolamine (0.2 # C i / m l ) . After washing the cells with PBS, the lipids were extracted, evaporated to dryness and analyzed by two-step, one-dimensional thin-layer chromatography and autoradiography as described under Experimental Procedures. Radioactive spots of plasmenylethanolamine and phosphatidylethanolamine were scraped off and counted. Data are expressed as cpm per 1.106 cells, and each value is the average + S.D. of three determinations.

4+ 18-t9+ 11+

1 3 1 2

2990+ 3140+ 3330+ 3 020 +

310 800 580 880

6 4 1 3 0 + 5420 5 7 3 6 0 + 6300 6 0 7 4 0 + 8180 61030-I- 13 490

The cloned P9OH R mutants were also found to be deficient in their content of plasmalogens. The level of plasmenylethanolamine was assessed by labeling with [2-14C]ethanolamine [7]. Table III shows that the plasmenylethanolamine content of wild-type cells was more than 11-fold that of the cloned P9OH R cell mutants. In addition, the incorporation of [2-14C]ethanolamine into the phosphatidylethanolamine of the cloned P9OH R mutants was increased almost 2-fold compared to wildtype, as in the mutants described previously [6,7].

Analysis of P90H and P9 uptake by flow cytometry The hypersensitivity of wild-type cells to P 9 O H / U V killing suggested that the amount of P9OH taken up a n d / o r incorporated into cell lipids would be higher in those cells than in peroxisome-deficient mutants. The relative ability of CHO-K1 and ZR-82 to accumulate P9OH was thus evaluated by flow cytometry. As shown in Fig. 2, CHO-K1 accumulates more P9OH than ZR-82. Recalculation from each model channel number indicated a 1.9-fold difference between the two cell lines (18 h incubation + 6 h wash), but only a 1.4-fold difference when the 6 hour wash was omitted. In contrast, CHO-K1 took up 1.7 to 1.9-times less P9 than ZR-82, consistent with previous work using 12-(l'-pyrene)dodecanoic acid, another pyrene fatty acid [23]. These data suggest that the alcohol function on the aliphatic chain (not the pyrene ring) is responsible for the reduced utilization of P9OH by the mutant cells.

Incorporation of P90H into lipids of CHO-K1 and ZR-82 The rather small differences in the incorporation of P9OH in wild-type and mutant cells (1.9-fold) seemed unlikely to account for the greater UV sensitivity (Fig. 1) of wild-type cells (60% and < 1% survival, respec-

138

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24 " -

16 .

(18h)~ .

.

.

t'

.

)

.

Z R - 8 2 'l . '

O

tu (.9

~

. , b

CHO-K1

CHO-K1

~' '],' '/ ,

" -

I , ~, ' I Z R - 8 2

',

40 i i

24 .

PgOH (18h+6h wash)

ZR-82

~, ~',,~

I t~ (

o

I

5

),~ ,, '

10

~

P9 (18h+6h wash)~

b

CHO-K1

, CNO-K 1

~,i~ ~

,

•

15

20

25

f}

5

10

' i

~

ZR-82 /Fi ,,, ,

15

20

t h a n in C H O - K 1 indicates that fatty alcohols are m e t a b o l i z e d m o r e slowly in these cells. A f t e r t r e a t m e n t with P 9 O H for 18 h a n d reincubation w i t h o u t P 9 O H for 6 h (wash), w i l d - t y p e cells h a d i n c o r p o r a t e d a b o u t 4 n m o l of P 9 O H p e r mg p r o t e i n a n d m u t a n t cells o n l y 2.3 ( T a b l e IV) - almost all of it as esterified P9. T h e i n c o r p o r a t i o n of P 9 O H into the phosp h o l i p i d s of Z R - 8 2 versus C H O - K 1 (Table IV) was m o r e strikingly r e d u c e d (2.4- a n d 7.5-fold). It was n o t restricted to e t h a n o l a m i n e - c o n t a i n i n g p h o s p h o l i p i d s (7.5-fold), b u t was also o b s e r v e d with p h o s p h a t i d y l choline, p h o s p h a t i d y l s e r i n e a n d p h o s p h a t i d y l i n o s i t o l ( T a b l e IV). In a d d i t i o n , [2-anC]acetate h a d been incor-

25

C H A N N E L NUMBER x 10

Fig. 2. Flow cytometry analysis of P9OH and P9 labeled cells. Wild-type and ZR-82 mutant cells, grown in 100 mm diameter tissue culture dishes, were incubated with P9OH (10 ~tM). In one experiment cells were harvested after 18 h. In another experiment cells were preincubated for 18 h in the presence of the fluorescent probe, then washed and reincubated in medium lacking probe for 6 h. At the end of each incubation, cells were washed with PBS and fixed in PBSparaformaldehyde. P9OH and P9 uptake by CHO-K1 and ZR-82 cells was measured by flow cytometry as described in Experimental Procedures. Fluorescence intensities varied within a range of 256 channels that constitute a logarithmic representation of three decades of fluorescence intensity. The modal channel was obtained by statistical analysis of the histogram.

tively, for Z R - 8 2 a n d C H O - K 1 , at 1 0 / ~ M P9OH). This o b s e r v a t i o n p r o m p t e d us to q u a n t i t a t e the i n c o r p o r a tion of P 9 O H into the different lipid species. T w o sets each of w i l d - t y p e a n d p e r o x i s o m e - d e f i c i e n t m u t a n t cells were i n c u b a t e d for 18 h at 3 7 ° C in the presence of 10 /~M P 9 O H . O n e set of C H O - K 1 a n d Z R - 8 2 was a n a l y z e d i m m e d i a t e l y to d e t e r m i n e the p a t t e r n of i n c o r p o r a t i o n of the fluorescent p r o b e into lipids. T h e o t h e r set was r e i n c u b a t e d for 6 h in the a b s e n c e of P 9 O H a n d then analyzed. Fig. 3 shows that the P 9 O H was i n c o r p o r a t e d into several distinct lipid species. T h e p h o s p h o l i p i d s ( l o c a t e d at the origin in Fig. 3 a n d consisting p r i m a r i l y of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin), as well as several a p o l a r lipids ( b a n d s 4, 5, 6 a n d 7), were less fluorescent in Z R - 8 2 t h a n in C H O - K 1 cells. T h e a c c u m u l a t i o n of u n m e t a b o l i z e d P 9 O H ( b a n d 3 in Fig. 3) in Z R - 8 2 after 18 h was m u c h greater than in C H O - K 1 . R e i n c u b a t i o n for 6 h in the absence of P 9 O H resulted in n e a r l y c o m p l e t e d i s a p p e a r a n c e of the free P 9 O H in b o t h C H O - K 1 a n d ZR-82. This is consistent with the results of Spector a n d S o b o r o f f [28], who showed that a significant a m o u n t of radioactive h e x a d e c a n o l is taken up b y c u l t u r e d cells a n d accumulates in the p l a s m a m e m b r a n e w i t h o u t b e i n g metabolized. The fact that u n m e t a b o l i z e d P 9 O H accumulates to a greater extent in p e r o x i s o m e - d e f i c i e n t cells

Fig. 3. Incorporation of P9OH into cellular lipids. Wild-type and ZR-82 mutant cells, grown in 100 mm diameter tissue culture dishes, were incubated with P9OH (10 /~M). In one experiment, cells were harvested after 18 h of incubation, and washed in PBS as described under Experimental Procedures. Lipids were extracted and analyzed by thin-layer chromatography using n-hexane/diethyl ether/acetic acid (60:40:1, v/v). In another experiment cells were incubated for 18 h in the presence of the fluorescent probe, then washed and reincubated in medium lacking P9OH for 6 h prior to lipid extraction and thin-layer chromatography. The silica gel plate, showing fluorescent lipids, was photographed as described under Experimental Procedures. The numbers are: 1, phospholipids (origin); 2, unidentified; 3, P9OH; 4, unidentified; 5, alkyl-diacylglycerol; 6, unidentified; 7, chloresteryl-pyrenenonanoate. Band 5 was tentatively identified as alkyl diacylglycerol based on its markedly reduced fluorescence in mutant ZR-82 and its relative R v. Band 7 was identified as cholesteryl-9-(l'-pyrene)nonanoateas described in Experimental Procedures.

139 TABLE IV

Incorporation of pyrene-labeled moieties of P90H into phospholipids and neutral lipids of wild-type and mutant cells Wild-type and ZR-82 mutant cells grown in 25 cm2 tissue culture flasks were labeled with P9OH (10 /~M) for 18 h followed by 6 h reincubation in the absence of P9OH. Next, the medium was removed, and the cells were harvested and washed with PBS prior to protein determination and lipid extraction. Phospholipids and neutral lipids were separated and identified by thin-layer chromatography. Each fluorescent lipid species was reextracted and its fluorescence intensity was measured as described in Experimental Procedures. Each value represents the amount of pyrene-labeled moieties incorporated (pmol) per mg protein and is the average+S.D, of three determinations. Lipid

CHO-K1 (pmol pyrene/mg protein)

ZR-82 (pmol pyrene/mg protein)

K1/82

Sphingomyelin Phosphatidylcholine Phosphatidylinositol +phosphatidylserine Phosphatidylethanolamine + plasmenylethanolamine

283 + 121 651 + 90

66 5-26 274 + 17

4.3 2.4

18+ 14

35- 2

6.0

294 + 35

39 5-17

7.5

P9OH Alkyl-diacylglyerola Cholesteryl-pyrenenonanoate Unidentified lipids b

715- 6 73 + 9 2519+ 192 84+ 11

1205- 8 19 + 9 1760+ 56 87 5- 17

0.6 3.8 1.4 0.9

Total

3 994 + 474

2 369 5-96

1.7

of Fig. 3. b Unidentified lipids include bands 2, 4 and 6 of Figure 3. a Band 5

porated into the cellular lipids prior to a n d d u r i n g the i n c u b a t i o n with P9OH, d e m o n s t r a t i n g that the composition of phospholipids a n d n e u t r a l lipids was n o t

affected b y P 9 O H in C H O - K 1 a n d Z R - 8 2 (data not shown). Presumably, the r e d u c t i o n of the i n c o r p o r a t i o n of P 9 O H into the p h o s p h o l i p i d s of m u t a n t Z R - 8 2 is the critical factor responsible for the increased resistance of P 9 O H - l a b e l e d Z R - 8 2 cells to long wavelength U V light.

Identification o f band 7 as cholesteryl-pyrenenonanoate A rapidly migrating, fluorescent c o m p o u n d was f o u n d in b o t h C H O - K 1 a n d Z R - 8 2 treated with P 9 O H ( b a n d 7, Fig. 3). Lipids extracted from cells i n c u b a t e d s i m u l t a n e o u s l y with [4-14C]cholesterol a n d P 9 O H (see E x p e r i m e n t a l Procedures) were analyzed b y thin-layer c h r o m a t o g r a p h y a n d autoradiography. Three radioactive b a n d s were observed, c o r r e s p o n d i n g respectively to free cholesterol, b a n d 7 a n d cholesterol ester (data n o t shown), i n d i c a t i n g that b a n d 7 did i n c o r p o r a t e cholesterol. B a n d 7 also migrated with s t a n d a r d cholesteryl-10-(l'-pyrene)decanoate, the fatty chain of which is only o n e c a r b o n a t o m larger t h a n the alkyl chain of P9OH. (Cells i n c u b a t e d in the presence of radioactive cholesterol together with hexadecanol or palmitic acid showed only radiolabeled cholesterol a n d cholesterol ester, b u t n o t b a n d 7). Next, b a n d 7 was extracted from the silica gel a n d exposed to alkaline a n d acidic hydrolysis (see E x p e r i m e n t a l Procedures). Acidic t r e a t m e n t failed to hydrolyze b a n d 7 a n d s t a n d a r d cholesteryl-10-(l'-pyrene)decanoate. O n the other h a n d , b o t h b a n d 7 a n d s t a n d a r d cholesteryl-10-(l'-pyrene)dec a n o a t e were completely hydrolyzed u p o n alkaline treatment. These results strongly suggest that b a n d 7 is a p y r e n e n o n a n o a t e ester of cholesterol, p r e s u m a b l y produced b y b o t h C H O - K 1 a n d Z R - 8 2 u p o n uptake a n d oxidation of P 9 O H to 9 - ( l ' - p y r e n e ) n o n a n o i c acid. P9-

TABLE V

Incorporation of [U-14C]hexadecanol into phospholipids and neutral lipids of wild-type and mutant cells Wild-type and mutant cells grown in sterile glass tubes were exposed to 10/~M [u-t4C]hexadecanol (2 #Ci/ml) in growth medium. In one set of tubes, the cells were incubated for 18 h, and then washed. Their lipids were extracted, evaporated to dryness, and analyzed by thin-layer chromatography and autoradiography, as described under Experimental Procedures. In another set of tubes, the cells were incubated for 18 h in the presence of 10/LM radiolabeled hexadecanol, then washed and reincubated in medium for 6 h, prior to final wash and lipid extraction. Radioactive bands corresponding to different lipids were scraped off and counted. Each value represents the amount of 14C incorporated (cpm) per/tg protein and is the average 5-S.D. of three determinations. Lipid

18 h with [U-14C]hexadecanol (cpm//~g protein)

18 h with [u-lac]hexadecanol + 6 h reincubation (cpm//~g protein)

CHO-K1

ZR-82

CHO-K1

Sphingomyelin Phosphatidylcholine Phosphatidylinositol+ phosphatidylserine Phosphatidylethanolamine+plasmenylethanolamine Fatty alcohol Fatty acid Triacylglycerol Alkyl-diacylglycerol Cholesterol ester

366 5- 21 2867 + 155 300 5- 27 26325-313 956 5- 53 405- 3 2685- 30 542 + 43 1435- 12

386 + 44 1193 + 144 201 5- 25 1795- 35 1314 5-109 195- 6 243+ 13 7 5- 1 219+ 17

427 + 9 2591 + 98 340 5- 14 28935-123 86 5- 26 265- 0.1 102+ 6 212 5- 8 505- 1

Total

8115 + 627

3 788 + 350

6727 + 197

ZR-82 387 5- 78 945 + 144 210 5- 34 2915- 58 97 5- 25 65- 1 975- 9 5 5- 1 875- 11 2125 5-356

140 treated cells (data not shown) also accumulated band 7, but the overall incorporation of P9 into phospholipids plus neutral lipids was higher in the peroxisome-deficient mutant cells than in the wild type.

Incorporation of [U-14C]hexadecanol into lipids of CHOK1 and ZR-82 Cells were incubated with [U-14C]hexadecanol under conditions similar to those employed with P9OH. The total incorporation of [U-14C]hexadecanol was 2-fold higher in CHO-K1 than in ZR-82 after an 18 h incubation. The reduced incorporation of [U-14C]hexadecanol in ZR-82 affected most phospholipids and apolar lipids, but was especially striking in the case of plasmenylethanolamine and alkyl diacylglycerides (Table V). The incorporation of [U-14C]hexadecanol into sphingomyelin was not reduced in ZR-82, an observation that is difficult to explain based on known biosynthetic pathways [29,30]. As in the case of P9OH, unmetabolized [U-14C]hexadecanol accumulated both in CHO-K1 and ZR-82 after the initial 18 h (Table V), but it disappeared after 6 h reincubation of the cells in the absence of [U-14C]hexadecanol. In contrast to cells treated with P9OH or P9, incubation of the CHO cells with [Ut4C]hexadecanol was not accompanied by the accumulation of excess radiolabeled cholesterol ester, analogous to the massive build-up of band 7 (Fig. 3).

Discussion The viability of peroxisome-deficient mutant cells labeled with P9OH and exposed to long wavelength UV light is several orders of magnitude higher than the viability of wild-type cells treated under similar conditions (Fig. 1). Although the true mechanism underlying this observation is not fully understood, this paper provides new insights into the metabolism of fatty alcohols by these mutants and also shows that this phenotype is a very useful tool for studying the genetics of peroxisome assembly. Because fatty alcohols are utilized by cells to synthesize plasmalogens [31-33], we initially assumed that a fluorescent fatty alcohol such as P9OH would be incorporated into the plasmalogens of wild-type cells, rendering those cells hypersensitive to photosensitization, whereas plasmalogen-deficient cells would be resistant. Although our hypothesis of an increased resistance of mutant ZR-82 against P 9 O H / U V treatment was confirmed, we observed that the incorporation of the fluorescent fatty alcohol was reduced in all phospholipids. Apparently, peroxisome-deficient cells are more resistant to P 9 O H / U V treatment not only because they are unable to make plasmalogens, but also because other routes of P9OH incorporation are reduced. The incorporation of the P9OH probe into ester-linked phospholipids of CHO cells shows that the derivative is,

in fact, converted to 9-(l'-pyrene)nonanoic acid in vivo, which is a substrate for phospholipid acylation. The utilization of [U-14C]hexadecanol, like P9OH, is also reduced in mutant ZR-82 (Table V), indicating that the pyrene ring is not responsible for the reduced incorporation of P9OH but, rather, that all fatty alcohols are metabolized more slowly in peroxisome/plasmalogendeficient cells than in wild type. Possibilities to account for the reduced incorporation of exogenous fatty alcohols include: (i) an elevated endogenous pool of unmetabolized fatty alcohols in the mutants; (ii) a partial reduction of a specific CoA-ligase a n d / o r glycerol-3phosphate acyltransferase in the mutants; or (iii) a partial reduction of the conversion of exogenous fatty alcohols to fatty acids in the mutants. A partial reduction of the enzymatic conversion of fatty alcohols to fatty acids is an attractive hypothesis, but we have not been able to demonstrate a defect in fatty alcohol: N A D + oxidoreductase [34-37] in extracts of ZR-82 (Morand, O., unpublished results). Interestingly, both P9 and P9OH-labeled cells accumulated a large amount of cholesteryl-9-(l'-pyrene)nonanoate (band 7, Fig. 3). The accumulation of excess cholesterol ester is not observed in cells grown in the presence of hexadecanol. Mutant ZR-82 is much more resistant to P 9 O H / U V even though it accumulated large amounts of these pyrene-containing apolar lipids. This suggests that the 2.4- to 7.5-fold reduction of incorporation of the probe into various phospholipids (Table IV) is responsible for the increased resistance of UV irradiation. In other words, essential membrane targets would be out of reach for the reactive oxygen species generated in pyrene-containing neutral lipid droplets, suggesting that cellular compartments must be considered in explaining the phenotypic effects of specific oxidative stresses. All the new mutants isolated so far (Tables I - I I I ) are peroxisome-deficient, just like the original strains isolated by Zoeller and Raetz [6]. By varying the exposure to P9OH and subjecting the cells to only one round of P 9 O H / U V treatment, it may be possible to isolate other mutations, such as those specifically defective in plasmalogen biosynthesis or fatty alcohol oxidation. The P 9 O H / U V method is also applicable to other cell lines, as was recently demonstrated by Zoeller [38] with macrophage tumor cells.

Acknowledgements This research was supported by National Institutes of Health grant D K 21722 (to C.R.H.R.). O.H.M. was on leave from Laboratoire de Neurochimie, Institut National de la Sant6 et de la Recherche Medicale Unit6 134, Paris, France. We would like to thank Eric Hanson for his expert assistance with the flow cytometry.

141

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19 20

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