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Isolation and Characterization of Peroxisome-Deficient Chinese Hamster Ovary Cell Mutants Representing Human Complementation Group III
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
233, 11–20 (1997)
EX973552
Isolation and Characterization of Peroxisome-Deficient Chinese Hamster Ovary Cell Mutants Representing Human Complementation Group III Kanji Okumoto, Akemi Bogaki,* Keita Tateishi, Toshiro Tsukamoto,* Takashi Osumi,* Nobuyuki Shimozawa,† Yasuyuki Suzuki,† Tadao Orii,†,1 and Yukio Fujiki2 Department of Biology, Kyushu University Faculty of Science, Fukuoka 812-81, Japan; *Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo 678-12, Japan; and †Department of Pediatrics, Gifu University, Gifu 500, Japan
tions in essential metabolic pathways, including b-oxidation of very long fatty acid and synthesis of etherglycerolipids such as plasmalogen [1]. Human peroxisomedeficient disorders are autosomal recessive [2] and include at least 10 different known genotypes [3–7], with three distinct phenotypes, manifested in cerebrohepatorenal Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease [1, 2]. To investigate mechanisms involved in peroxisome biogenesis and the primary defects of human peroxisome assembly disorders, we earlier isolated three mutually distinct peroxisome-deficient Chinese hamster ovary (CHO)3 cell mutants, Z24, Z65, and ZP92, by colony-autoradiographic screening and the 9-(1*-pyrene)nonanol/ultraviolet (P9OH/UV) selection methods [6, 8]. The mutant cells resembled fibroblasts from patients with peroxisome-deficient disease such as Zellweger syndrome, with defects in peroxisome assembly, but the synthesis of peroxisomal proteins was normal. Complementation groups between three CHO mutants and nine groups of fibroblasts from patients with peroxisome-deficient disorders were defined [6, 9]. Peroxisome assembly factor-1 (PAF-1, termed Pex2p by a unified nomenclature [10]), a 35-kDa peroxisomal integral membrane protein, proved to be essential for restoration of peroxisome biogenesis in the CHO mutant Z65 [11]. The PEX2 gene was shown to be the first pathogenic gene of Zellweger syndrome; a homozygous nonsense mutation (Arg119Stop) was the genetic cause of this syndrome in a complementation group F patient in Japan (the same as X in the United States and 5 in
We made use of the 9-(1*-pyrene)nonanol/ultraviolet (P9OH/UV) method and isolated peroxisome-deficient mutant cells. TKa cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1) were used to avoid frequent isolation of the Z65-type, PEX2-defective mutants. P9OH/UV-resistant cell colonies were examined for the intracellular location of catalase, a peroxisomal matrix enzyme, by immunofluorescence microscopy and using anti-catalase antibody. As six mutant cell clones showed cytosolic catalase, there was likely to be a deficiency in peroxisome assembly. These mutants also showed the typical peroxisome assembly-defective phenotype, including significant decrease of dihydroxyacetonephosphate acyltransferase, the first step key enzyme in plasmalogen synthesis, and loss of resistance to 12-(1*-pyrene)dodecanoic acid/UV treatment. By transfection of Pex2p and Pex6p (formerly PAF-2) cDNAs and cell fusion analysis between the CHO cell mutants, two mutants, ZP104 and ZP109, were found to belong to a novel complementation group. Further complementation analysis using fibroblasts from patients with peroxisome biogenesis disorders revealed that the mutants belonged to human complementation group III. Taken together, ZP104 and ZP109 are in a newly identified fifth complementation group in CHO mutants reported to date and represent the human complementation group III. q 1997 Academic Press
INTRODUCTION
Peroxisome is a single membrane-bounded, ubiquitous intracellular organelle in eukaryotes and func-
3
Abbreviations used: AOx, acyl-CoA oxidase; CHO, Chinese hamster ovary; DHAP-ATase, dihydroxyacetonephosphate acyltransferase; FCS, fetal calf serum; Hygr, resistant to hygromycin; PMP70, 70-kDa peroxisomal integral membrane protein; Ouar, resistant to ouabain; P9OH/UV, 9-(1*-pyrene) nonanol/ultraviolet; P12, 12-(1*pyrene)dodecanoicacid; PAF-1 and PAF-2, peroxisome assembly factor-1 and -2, respectively; PTS1R, peroxisome targeting signal type 1 receptor; TGr, resistant to 6-thioguanine.
1
Present address: Chubu Women’s College, Seki, Japan. To whom correspondence and reprint requests should be addressed at Department of Biology, Kyushu University Faculty of Science, 6-10-1 Hakozaki, Fukuoka 812-81, Japan. Fax: (092) 6422645. E-mail: [email protected]. 2
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0014-4827/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Immunofluorescent staining of wild-type CHO cells, mutants, and hybrid cells. (A) Cells were incubated with rabbit anti-rat catalase antibody. (a) Wild-type CHO cells; (b) PEX2-transformed wild-type, TKa; (c–h) mutants ZP104, ZP105, ZP106, ZP107, ZP108, and ZP109, respectively. Scale, 30 mm. (B) Cells were stained with anti-rat PMP70 antibody. (a) TKa; (b) ZP104; (c) ZP109. Scale, 20 mm.
Europe) [9, 12]. Recently, peroxisome assembly factor-2 (PAF-2) cDNA, termed PEX6, that restored peroxisome assembly in the mutant ZP92 as well as the fibroblasts derived from patients with Zellweger syndrome of complementation group C in Japan (the same as group IV in the United States and group 3 in Europe) was cloned [13, 14] and was found to encode a member of the putative ATPase (AAA) family [15]. Several other complementing genes of yeast mutants such as Saccharomyces cerevisiae PAS1 (PEX1), PEX6 from Pichia pastoris (formerly PAS5), and Yallowia lipolytica (formerly PAY4) also belong to this family [16–18]. Thus, peroxisome assembly-defective CHO cell mutants are indeed a useful system for the study of peroxisome biogenesis
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and for elucidating primary defects of human peroxisome disorders. We report here a new complementation group of CHO cell mutants that apparently belongs to the human complementation group III. MATERIALS AND METHODS Selection of peroxisome-deficient CHO cell mutants. Wild-type CHO-K1 cells were cultured in Ham’s F12 medium supplemented with 10% fetal calf serum (FCS) and transfected with rat Pex2p cDNA, a plasmid pcD65K [11], as described. Stable transformant, TKa, selected with G418, was found to express rat PEX2 mRNA [19]. TKa cells were treated with N-methyl-N*-nitro-N-nitrosoguanidine (Nacalai Tesque, Kyoto, Japan) and grown in F12 medium containing
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FIG. 2. Complementation analysis by cDNA transfection. CHO cell mutants ZP106 and ZP109 were each transfected with cDNA coding for Pex2p or Pex6p (formerly PAF-1 and PAF-2, respectively) and stained with anti-catalase antibody. (a and b) ZP106; (c and d) ZP109. Peroxisomes were noted only in b. Scale, 30 mm. 10% FCS for 24 h. The growth medium was changed to the F12containing 10% FCS and 6 mM P9OH (Molecular Probes, Inc., Eugene, OR) and the cells were further cultured for 20 h. The cells were washed once with F12 and incubated for 6 h in F12 with 10% FCS. After a thorough wash of the cells with F12, mutants deficient in peroxisomes were selected by a 2-min exposure to UV light, as described [6]. Assays. Dihydroxyacetonephosphate acyltransferase (DHAPATase) [20], the latency of catalase [6], and resistance to P9OH/ UV and 12-(1*-pyrene)dodecanoic acid (P12)/UV treatments [6] were measured, as described. P12 was from Molecular Probes, Inc. Morphological analysis. Peroxisomes in CHO cells were visualized by indirect immunofluorescence light microscopy using rabbit antibody to rat liver catalase and those in human fibroblasts were detected with rabbit anti-human catalase antibody, as described [6]. Antigen–antibody complex was detected by fluorescein isothiocyanate-labeled goat anti-rabbit immunogloblin G antibody (Cappel, Durham, NC; ZYMED Laboratories, Inc., South San Fransisco, CA) under a Carl Zeiss Axioskop FL microscope (Oberkochen, Germany). Several CHO mutant cell clones were stained with rabbit antibody to 70-kDa peroxisomal integral membrane protein (PMP70) [6]. DNA transfection. DNA transfection to CHO mutant cells was done with plasmids pcD65K and pUcD2r92A, mammalian expression vectors containing cDNAs for rat Pex2p and Pex6p, respectively [11, 13]. Two methods were used: one was electroporation [13], except that cells suspended in phosphate-buffered saline (magnesium- and
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calcium-free) at 1 1 106 cells/ml were mixed with 10 mg plasmid DNA and electroporated using an electroporator, Model ECM600 (BTX Inc., San Diego, CA) on a setting of 300 V, 400 mF, and 72 V; the other method was liposome-mediated transfection [21] but with some modification. Briefly, cells that had been plated on a coverslip 1 day before transfection at 8 1 104 cells in a 35-mm dish were washed twice with Hepes-buffered saline (20 mM Hepes–KOH, 150 mM NaCl, pH7.4) supplemented with 1 mM CaCl2 and 0.5 mM MgCl2 . Three micrograms of plasmid DNA and 10 mg of cationic liposome (O,O*-ditetradecanolyl-N-(trimethylammonioacetyl) diethanolamine chloride; 14Dea2, a gift from A. Ito) prepared by sonication were separately diluted with 300 ml of Hepes-buffered saline containing CaCl2 and MgCl2 . Both solutions were mixed and poured over the cells. After incubation for 1 h at 377C, the DNA–liposome mixture was removed from the culture medium by aspiration and fresh F12 medium with 10% FCS was added. The cells were cultured for 2 days and then were further incubated overnight in 2 ml of serum-free F12 medium before immunostaining. Cell fusion and labeling of cell protein. We used variants of cell mutants resistant to 6-thioguanine (TGr) plus ouabain (TGrOuar) [8]. CHO cells resistant to 6-thioguanine and hygromycin (TGrHygr) were isolated by transfection of a plasmid pMiw hph (a gift from H. Kondo) to TGr variants, using the calcium phosphate method [22] followed by selection in the presence of 200 U/ml hygromycin B (Wako, Osaka, Japan). Parent CHO cells and the CHO cells to be examined were cocultured for 24 h and then fused with the use of polyethylene glycol,
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TABLE 1 Complementation Group Analysis of CHO Cell Mutants by cDNA Transfection Transfection of cDNA coding for CHO mutant ZP104 ZP105 ZP106 ZP107 ZP108 ZP109 Z65 ZP92
Pex2p
Pex6p
0 0 0 0 0 0 / 0
0 0 / 0 0 0 0 /
Note. Cells were stained with anti-rat catalase antibody 3 days after cDNA transfection. (/) Peroxisomes were complemented; (0) not complemented.
as described [6], except that selection of fused cells was carried out with 1 mM ouabain or 200 U/ml hygromycin B (Wako, Osaka, Japan). Cell fusion of CHO mutant TGr variants with human fibroblasts was done as described [6]. Metabolic labeling of cells with 20 mCi/ml of [35S]methionine and [35S]cysteine (New England Nuclear, Boston, MA) for 24 h in F12 medium and immunoprecipitation of peroxisomal proteins from cell lysates were done, as described [8].
RESULTS
Isolation and Morphological Analysis of CHO Cell Mutants PEX2-transformed CHO-K1, TKa cells were mutagenized and those cells resistant to P9OH/UV treatment were selected. Viable cell colonies were examined for peroxisome morphology, using an anti-catalase antibody. Six mutant cell clones, ZP104, ZP105, ZP106,
ZP107, ZP108, and ZP109, were isolated by cell cloning from mutant colonies with diffuse catalase present in the cytoplasm, with clear presence in the cytosol, and a phenotype similar to that previously isolated mutants, Z24, Z65, and ZP92 [6, 8] (Fig. 1A, c–h). In most P9OH/ UV-resistant cells as well as the wild-type CHO-K1 and TKa, catalase was detected in numerous particles, presumably peroxisomes (Fig. 1A, a and b). At present, we have no explanation as to why there was occasional resistance of some peroxisome-positive cells to the P9OH/UV treatment. Mutants ZP104 and ZP109 were then stained with antiserum against PMP70 (Fig. 1B, b and c). Larger but fewer particles immunoreactive with anti-70 IMP antibody were detected, consistent with findings in previously isolated CHO cell mutants [6] and similar to ‘‘peroxisomal ghost’’ vesicles in the fibroblasts from Zellweger patients [23–25]. These particles were also noted in other mutants, ZP105 to ZP108 (not shown). There was a punctate staining pattern in the wild-type cells, as seen with anti-catalase antibody (Fig. 1B, a). Complementation Group Analysis by cDNA Transfection Mutant cells, ZP104 to ZP109, were each transfected with cDNA for Pex2p or Pex6p. Transient transfectants were analyzed by indirect immunofluorescence microscopy using anti-catalase antibody. Numerous peroxisomes were noted only in ZP106 transfected with Pex6p cDNA, thereby indicating that ZP106 belongs to the complementation group of ZP92, whereas all of the other transfectants showed cytosolic catalase (Fig. 2 and Table 1). As a control, peroxisome assembly in Z65 cells was restored by Pex2p cDNA, but not with Pex6p cDNA, consistent with earlier findings [11]. Conversely, peroxisome formation in ZP92 cells was com-
FIG. 3. Cell fusion analysis of CHO cell mutants. CHO cell mutants ZP104 and ZP105 were fused with ZP109 and stained with antirat catalase antibody. (a) Fusion of ZP104 with ZP109; (b) ZP105 with ZP109. Scale, 20 mm.
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TABLE 2 Complementation Group Analysis of CHO Mutants by Cell Fusion
ZP104 ZP105 ZP107 ZP108 ZP109
ZP104
ZP105
ZP107
ZP108
ZP109
N.D. / / / 0
/ N.D. / / /
/ / N.D. 0 /
/ / 0 N.D. /
0 / / / 0
fused cells of each mutant with the wild-type CHO-K1 showed catalase-positive particles as numerous as in the wild type, suggesting that a lesion(s) of the allele(s) in the mutants are recessive (Table 3). The mutant ZP109 was then used for further analyses. Latency of Catalase
plemented only with Pex6p and not by Pex2p expression [13]. Accordingly, the CHO mutants isolated in this study, except for ZP106, belong to a complementation group(s) different from those of Z65 and ZP92. Moreover, no Z65-type mutant was isolated, thereby clearly demonstrating the efficacy of TKa cells in isolating mutants devoid of PEX2 deficiency.
The cellular location of catalase was also examined by the digitonin assay [6, 8]. At 100 mg/ml of digitonin, full activity of catalase was detected in the mutant ZP109 as in Z65, whereas Ç60% of the activity was latent in the wild-type cells, CHO-K1 and TKa (Fig. 5). Mutants ZP104, ZP105, ZP106, ZP107, and ZP108 also showed catalase latency similar to that of ZP109 (data not shown). Treatment with 300 mg/ml of digitonin released full catalase activity of the wild type. These results were interpreted to mean that catalase is localized in the cytosol in these mutants. Cell hybrids of ZP109 and Z65 showed the same profile of catalase latency as the parent TKa and CHO-K1, strongly suggesting that these mutants differ in the complementation group, consistent with observations in the cell fusion study and the morphological analysis.
Cell Fusion Analysis
Cell Fusion with Fibroblasts from Zellweger Patients
To classify six CHO mutants, ZP104 to ZP109, all combinations of cell fusion excluding ZP106 were set up (Fig. 3, Table 2). Fusion of ZP104 with ZP109 and ZP107 with ZP108 produced no peroxisomal punctate structure, indicating that the respective pair of mutants belong to the same complementation group (Fig. 3a, Table 2). Peroxisomes were noted in hybrid cells between ZP105 and ZP109, which means that ZP105 is distinct from ZP109 (Fig. 3b). Likewise, peroxisomes were complemented in all of the other fused cells, except for a homologous hybrid of ZP109 (Table 2). Accordingly, the mutants isolated in this study could be classified into four complementation groups, i.e., ZP104 and ZP109 are in one group; ZP105, ZP106, and ZP107, as well as ZP108 are, respectively, in three different groups. Next, the CHO mutants were fused with previously isolated Z24 and ZP102 (the same complementation group as human group II in the United States [19]). Peroxisomes were not complemented in assembly by cell fusion between ZP105 and ZP102, ZP107 and Z24, and ZP108 and Z24, while other cell hybrids such as those of ZP109 with Z65, ZP92, Z24, and ZP102 did have peroxisome particles (Fig. 4, Table 3). These observations strongly suggest that ZP104 and ZP109 belong to a novel complementation group of CHO mutants and that ZP107 and ZP108 are in the same complementation group as Z24. ZP105 belongs to complementation group ZP102, the same as human group II. Moreover,
To determine to which human complementation group the CHO mutant ZP109 belonged, cell fusion was carried out between ZP109 and human fibroblasts from peroxisome-deficient patients with Zellweger syndrome; complementing genes were not delineated. Peroxisomes were not complemented in fused cells of ZP109 and fibroblasts from a Zellweger patient of complementation group III, whereas numerous punctate catalase-containing structures, peroxisomes, were noted in the hybrid cells of ZP109 and fibroblasts from a patient of complementation group B in Japan (the same as group VII in the United States) (Fig. 6). Like-
Note. Cell fusion was done as under Materials and Methods. Unselected fused cells were stained with anti-rat catalase antibody. (/) Peroxisomes were complemented; (0) not complemented; N.D., not done.
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TABLE 3 Complementation Group Analysis of CHO Mutants by Cell Fusion
ZP104 ZP105 ZP107 ZP108 ZP109
Z65a
ZP92a
Z24a
ZP102b
CHO-K1a
N.D. N.D. N.D. N.D. /
N.D. N.D. N.D. N.D. /
/ / 0 0 /
/ 0 / / /
/ / / / /
Note. Cell fusion was done as for Table 2. Fused cells were selected and stained with anti-rat catalase antibody. (/) Peroxisomes were complemented; (0) not complemented; N.D., not done. a,b Cells resistant to 6-thioguanine plus ouabain [6, 8] and 6-thioguanine plus hygromycin, respectively.
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FIG. 4. Complementation group analysis of CHO cell mutants. Mutants were pairwise fused and after selection, the hybrids were stained with anti-catalase antibody. (a–c) Fused cells of ZP105 with ZP102TGrHygr, ZP107 with Z24TGrOuar, and ZP108 with Z24TGrOuar, respectively; (d–h) hybrid cells of ZP109 with Z65TGrOuar, ZP92TGrOuar, Z24TGrOuar, ZP102TGrHygr, and the wild-type CHO-K1TGrOuar, respectively. Scale, 20 mm.
wise, peroxisomes were noted in fused cells between ZP109 and peroxisome-deficient patient fibroblasts of groups A (the same as group VIII in the United States), D (the same as group IX), G [7], and VI, respectively (Table 4). Taken together, ZP109 apparently belongs to human complementation group III, distinct from groups A, B, D, G, and VI. Properties of CHO Cell Mutants Mutants ZP105 and ZP109 were severely defective in DHAP-ATase activity and were sensitive to P12/UV
FIG. 5. Latency of catalase in wild-type, mutant, and hybrid cells. Latency of catalase was determined as described [8]. (s) Wildtype CHO-K1; (n), CHO-TKa; (l), ZP109; (m), Z65; (h), ZP109 1 Z65TGrOuar. The results were represented as averages of duplicate assays.
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treatment as was noted for mutants Z24, Z65, and ZP92 [6, 8], while the DHAP-ATase level in TKa cells was as high as that of the wild-type CHO-K1 (Table 5). DHAP-ATase activity of other mutants, ZP104, ZP106, and ZP107, was also low (not shown). After cell culture in the presence of P9OH followed by a short exposure to UV, over 90% of ZP105 and ZP109 cells survived, but only less than 0.001% of TKa cells were viable, in good agreement with the previous findings for the wildtype CHO-K1 and mutants Z24, Z65, and ZP92 [6, 8]. The mutant cells were highly sensitive to P12/UV treatment, but nearly 70% of TKa cells were resistant, again consistent with previous deductions [8]. These cell-phenotypic properties were also noted for the other mutants, ZP104, ZP106, ZP107, and ZP108 (not shown). Biogenesis of peroxisomal enzymes was investigated in the mutant ZP109 and its hybrid with Z65 that had been labeled with [35S]methionine and [35S]cysteine. Acyl-CoA oxidase (AOx), the first enzyme of the peroxisomal b-oxidation system, is a heterodimer comprising 75-kDa A, 53-kDa B, and 22-kDa C polypeptide components [8, 26]; B and C are derived from A [27]. All three 35 S-labeled polypeptide components were evident in the wild-type CHO-K1 and TKa (Fig. 7, lanes 1 and 2) with a faint band of C attributed to the low content of methionine and cysteine [8, 26], whereas the reduced 75-kDa 35 S-labeled A form of AOx, but not converted forms (53-kDa B and 22-kDa C components), was detected in ZP109 and Z65 because of the rapid degradation of AOx-A (Fig. 7, lanes 3 and 4), as noted in the mutants Z24 and ZP92 [6, 8]. The third enzyme of the peroxi-
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FIG. 6. Cell fusion of ZP109 with fibroblasts from Zellweger patients. (a and b) Cell hybrids of ZP109 with fibroblasts from Zellweger patients of human complementation groups III and B, respectively. Note the numerous peroxisomes in b but not in a. Scale, 20 mm.
somal b-oxidation system, 3-ketoacyl-CoA thiolase (thiolase), is synthesized as a larger precursor of 44 kDa and then processed to a 41-kDa mature form [8]. Only a 44-kDa 35S-labeled precursor of thiolase was detectable in ZP109 and Z65, consistent with the finding in Z24 and ZP92 [6, 8], although the 41-kDa mature protein was apparent in the wild-type cells, CHO-K1 and TKa (Fig. 7, lanes 6–9). Thus, peroxisomal proteins are apparently synthesized at a normal level in mutants, as concluded from previous studies [6, 8]. In the hybrid of ZP109 with Z65, appropriate processing of AOx and thiolase was evident, as in the wild type (Fig. 7, lanes 5 and 10, respectively). These observations support the notion that the CHO mutant ZP109 belongs to a complementation group distinct from Z65. TABLE 4 Complementation Analysis of ZP109 by Cell Fusion with Fibroblasts from Patients with Peroxisome-Deficient Disorders Complementation groups of patient fibroblastsa
ZP109b
A
B
D
G
III
VI
/
/
/
/
0
/
Note. After cell fusion, fused cells were selected and stained with anti-human catalase antibody as described [6]. (/) Peroxisomes were complemented; (0) not complemented. a Used fibroblasts were from peroxisome-deficient patients belonging to complementation groups whose complementing genes were not identified. b ZP109 cells resistant to 6-thioguanine.
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DISCUSSION
A search for genes essential for peroxisome assembly has entailed significant efforts, as has elucidation of primary defects of peroxisome biogenesis disorders, including Zellweger syndrome and neonatal adrenoleukodystorophy. cDNAs coding for Pex2p and Pex6p were cloned by phenotypic complementation of CHO mutants Z65 and ZP92, respectively [11, 13]. Mutations in the PEX2 gene proved to be the cause of Zellweger syndrome of complementation group F (the same as groups X and 5) [9] (Table 6). Human cDNA encoding a receptor, PEX5, for peroxisome targeting signal type 1, SKL motif was independently isolated by an expressed sequence tag search with P. pastoris PAS8 [28], immunoscreening of a cDNA expression library [29], and a yeast two-hybrid system [30]. PEX5 and PEX6 were shown to be responsible for primary defects in the patients of complementation groups II and C (the same as group IV) disorders [13, 14, 28, 29, 31]. In the present work, we investigated by mutant selection peroxisome-deficient CHO cell mutants distinct from the previous Z24, Z65, and ZP92. By making a modification with use of the TKa, the wild-type expressing rat Pex2p mRNA [19], frequent isolation of Z65type mutants [6] was avoided. After several cycles of the mutant isolation procedure, i.e., P9OH/UV treatment followed by indirect immunofluorescence microscopy, six mutant cell clones were isolated, from which two, ZP104 and ZP109, were found to belong to a newly defined complementation group. All of the CHO cell
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TABLE 5 Characteristics of Peroxisome-Deficient CHO Cell Mutants CHO cell
Peroxisomes
Catalase latency (%)
DHAP-ATase (%)
P9OH/UV (%)
P12/UV (%)
Wild type TKa ZP105 ZP109
/ / 0 0
63 63 0 0
100 100 11 10
õ0.001a õ0.001 93 99
87a 66 õ0.001 õ0.001
Note. Catalase latency represents peroxisomal catalase, calculated as described [8]. Peroxisomal DHAP-ATase was measured as described [20]. Survival rates of P9OH/UV- and P12/UV-resistant cells were expressed as percentages of the unselected control [6]. a From reference [6].
mutants we isolated herein showed typical, common properties characterized for previously isolated Z24, Z65, and ZP92, including a recessive lesion(s), lacking morphologically recognizable peroxisomes, no latency of catalase, a severe loss of DHAP-ATase, and high sensitivity to the P12/UV treatment but with a normal synthesis of peroxisomal proteins. The mutants also contained peroxisomal ghost-like vesicular structures, as described for all of the CHO mutants [6] and for fibroblasts from patients with peroxisome-deficient dis-
FIG. 7. Biogenesis of peroxisomal proteins. Cells were labeled for 24 h with [35S]methionine and [35S]cysteine. Cell types are indicated at the top. Immunoprecipitation was done with rabbit antirat acyl-CoA oxidase (AOx) and 3-ketoacyl-CoA thiolase antibodies. Immunoprepicitates were analyzed by SDS–PAGE (12% polyacrylamide gel). Radioactive polypeptide bands were detected using a FujiX BAS1000 Bio-Imaging Analyzer (Fuji Photo Film, Tokyo, Japan). Exposure 20 h. Arrows show the position of AOx components; open and solid arrowheads indicate a larger precursor and mature protein of thiolase, respectively.
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ease [23–25]; however, the significance of the ‘‘peroxisomal ghost’’ is not clear. Accordingly, mutants isolated in this study represent typical somatic mammalian cell mutants defective in peroxisome biogenesis. By complementation analysis, six mutants were grouped into four (Table 6). Mutant ZP105 is in the same complementation group as ZP102 which belongs to human complementation group II [19], where the PTS1 receptor is responsible for the primary defect [28, 29]. Thus, ZP105 may serve as a model mammalian cell system in which to examine functions and dysfunctions of PTS1 receptor at molecular and cellular levels. ZP106, like ZP92, is in the human complementation group C (the same as groups IV and 3) in which malfunction of Pex6p is evident [13]. ZP107 and ZP108 belong to the complementation group Z24, human complementation group E (the same as groups 1 and 2). ZP104 and ZP109 were classified into the complementation group III. Accordingly, mutants ZP104 and ZP109 are in a newly identified, fifth complementation group of CHO cell mutant clones that belong to complementation group III, among 10 human complementation groups [3–7]. The import of both PTS1- and PTS2 proteins is defective in ZP109 (not shown), consistent with the results using fibroblasts of complementation group III patients [32]. Therefore, mutants ZP104/ ZP109 and ZP107/ZP108 will be useful for cloning genes essential for peroxisome biogenesis in complementation groups III and I (the same as groups E and 2) as well as for studies on mechanisms of peroxisome assembly in mammals. At this writing, only three responsible genes, PEX2 [9, 11, 33, 34], PEX5 [28, 29], and PEX6 [13, 14, 31] are elucidated. During the course of this study, P9OH/UV-resistant, viable cell colonies but with the peroxisomal punctate catalase-staining pattern were evident. Similar findings were reported for murine macrophage-like cell lines isolated using the same method [35]. Several explanations such as reduced incorporation of P9OH by mutation(s) can be given for the viability of peroxisome-
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TABLE 6 Complementation Groups and Complementing Genes of Peroxisome Deficiency Human fibroblasts Japan (Gifu)
U.S.A. (Kennedy)
A B C D E F
VIII VII IV IX I X II III VI
Europe (Amsterdam)
3 2 5 4
Gd
Phenotype ZS, NALD, IRD ZS, NALD ZS ZS ZS, NALD, IRD ZS ZS, NALD ZS NALD ZS
CHO mutants
ZP106, ZP92b
Complementing gene
PEX6 (PAF-2)
a
Z24, ZP107, ZP108 Z65a ZP102,c ZP105 ZP104, ZP109
PEX2 (PAF-1) PEX5
Note. ZS, Zellweger syndrome; NALD, neonatal adrenoleukodystrophy; IRD, infantile Refsum disease. a,b From refs. [6, 8]; c,dfrom [19, 7].
Cregg, J. M., Dodt, G., Fujiki, Y., Goodman, J. M., Just, W. W., Kiel, J. A. K. W., Kunau, W.-H., Lazarow, P. B., Mannaerts, G. P., Moser, H., Osumi, T., Rachubinski, R. A., Roscher, A., Subramani, S., Tabak, H. F., Tsukamoto, T., Valle, D., van der Klei, I., van Veldhoven, P. P., and Veenhuis, M. (1996) J. Cell Biol. 135, 1–3.
positive cells under the conditions used; therefore, further investigation is warranted. We thank H. W. Moser for patients’ fibroblasts of complementation groups III and VI, A. Ito for supply of liposomes, H. Kondo for the generous gift of a plasmid, K. Ohashi for technical advice, and K. Mizuno and M. Ohara for helpful comments. This work was supported in part by Grants-in-Aid for Scientific Research (07408016 to Y.F.) and by grants (to Y.F.) from Uehara Memorial Foundation, Mitsubishi Foundation, Nagase Science and Technology Foundation, Naito Foundation, and Ciba-Geigy Foundation (Japan) for the Promotion of Science.
REFERENCES 1. van den Bosch, H., Schutgens, R. B. H., Wanders, R. J. A., and Tager, J. M. (1992) Annu. Rev. Biochem. 61, 157–197. 2. Lazarow, P. B., and Moser, H. W. (1994) in The Metabolic Basis of Inherited Disease (Scriver, C. R., Beaudet, A. I., Sly, W. S., and Valle, D., Eds.), pp. 2287–2324, McGraw–Hill, New York. 3. Brul, S., Westerweld, 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) J. Clin. Invest. 81, 1710– 1715. 4. Roscher, A. A., Hoefler, S., Hoefler, G., Paschke, E., Paltauf, F., Moser, A., and Moser, H. (1989) Pediatr. Res. 26, 67–72. 5. Yajima, S., Suzuki, Y., Shimozawa, N., Yamaguchi, S., Orii, T., Fujiki, Y., Osumi, T., Hashimoto, T., and Moser, H. W. (1992) Hum. Genet. 88, 491–499. 6. Shimozawa, N., Tsukamoto, T., Suzuki, Y., Orii, T., and Fujiki, Y. (1992) J. Clin. Invest. 90, 1864–1870. 7. Poulos, A., Christodoulou, J., Chow, C. W., Goldblatt, J., Paton, B. C., Orii, T., Suzuki, Y., and Shimozawa, N. (1995) J. Pediatr. 127, 596–599. 8. Tsukamoto, T., Yokota, S., and Fujiki, Y. (1990) J. Cell. Biol. 110, 651–660. 9. Shimozawa, N., Tsukamoto, T., Suzuki, Y., Orii, T., Shirayoshi, Y., Mori, T., and Fujiki, Y. (1992) Science 255, 1132–1134. 10. Distel, B., Erdmann, R., Gould, S. J., Blobel, G., Crane, D. I.,
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11. Tsukamoto, T., Miura, S., and Fujiki, Y. (1991) Nature 350, 77– 81. 12. Shimozawa, N., Suzuki, Y., Orii, T., Moser, A., Moser, H. W., and Wander, R. J. A. (1993) Am. J. Hum. Genet. 52, 843–844. 13. Tsukamoto, T., Miura, S., Nakai, T., Yokota, S., Shimozawa, N., Suzuki, Y., Orii, T., Fujiki, Y., Sakai, F., Bogaki, A., Yasumo, H., and Osumi, T. (1995) Nature Genet. 11, 395–401. 14. Fukuda, S., Shimozawa, N., Suzuki, Y., Tomatsu, S., Tsukamoto, T., Hashiguchi, N., Osumi, T., Masuno, M., Imaizumi, K., Kuroki, Y., Fujiki, Y., Orii, T., and Kondo, N. (1996) Am. J. Hum. Genet. 59, 1210–1220. 15. Kunau, W. H., Beyer, A., Franken, T., Gotte, K., Marzioch, M., Saidowsky, J., Skaletz-Rorowski, A., and Wiebel, F. F. (1993) Biochimie 75, 209–224. 16. Erdmann, R., Wiebel, F. F., Flessau, A., Rytka, J., Beyer, A., Frohlich, K. U., and Kunau, W. H. (1991) Cell 64, 499–510. 17. Spong, A. P., and Subramani, S. (1993) J. Cell Biol. 123, 535– 548. 18. Nuttley, W. M., Brade, A. M., Eitzen, G. A., Veenhuis, M., Aitchison, J. D., Szilard, R. K., Glover, J. R., and Rachubinski, R. A. (1994) J. Biol. Chem. 269, 556–566. 19. Tsukamoto, T., Bogaki, A., Okumoto, K., Tateishil, K., Fujiki, Y., Shimozawa, N., Suzuki, Y., Kondo, N., and Osumi, T. (1997) Biochem. Biophys. Res. Commun. 230, 402–406. 20. Shimozawa, N., Suzuki, Y., Orii, T., Yokota, S., and Hashimoto, T. (1988) Pediatr. Res. 24, 723–727. 21. Akao, T., Nakayama, T., Takeshita, K., and Ito, A. (1994) Biochem. Mol. Biol. Int. 34, 915–920. 22. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745– 2752. 23. Wiemer, E. A. C., Brul, S., Just, W. W., van Driel, R., BrouwerKelder, E., van den Berg, M., Weijers, P. J., Schutgens, R. B. H.,
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27. Miyazawa, S., Osumi, T., Hashimoto, T., Ohno, K., Miura, S., and Fujiki, Y. (1989) Mol. Cell. Biol. 9, 83–91. 28. Dodt, G., Braverman, N., Wong, C. S., Moser, A., Moser, H. W., Watkins, P., Valle, D., and Gould, S. J. (1995) Nature Genet. 9, 115–125.
33. 34. 35.
29. Wiemer, E. A., Nuttley, W. M., Bertolaet, B. L., Li, X., Francke, Received July 15, 1996 Revised version received February 27, 1997
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U., Wheelock, M. J., Anne, U. K., Johnson, K. R., and Subramani, S. (1995) J. Cell Biol. 130, 51–65. Fransen, M., Brees, C., Baumgart, E., Vanhooren, J. C., Baes, M., Mannaerts, G. P., and Van Veldhoven, P. P. (1995) J. Biol. Chem. 270, 7731–7736. Yahraus, T., Braverman, N., Dodt, G., Kalish, J. E., Morrell, J. C., Moser, H. W., Valle, D., and Gould, S. J. (1996) EMBO J. 15, 2914–2923. Slawecki, M. L., Dodt, G., Steinberg, S., Moser, A. B., Moser, H. W., and Gould, S. J. (1995) J. Cell Sci. 108, 1817–1829. Thieringer, R., and Raetz, C. R. H. (1993) J. Biol. Chem. 268, 12631–12636. Tsukamoto, T., Shimozawa, N., and Fujiki, Y. (1994) Mol. Cell. Biol. 14, 5458–5465. Zoeller, R. A., Rangaswamy, S., Herscovitz, H., Rizzo, W. B., Hajra, A. K., Das, A. K., Moser, H. W., Moser, A., Lazarow, P. B., and Santos, M. J. (1992) J. Biol. Chem. 267, 8299–8306.
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EX973552
Isolation and Characterization of Peroxisome-Deficient Chinese Hamster Ovary Cell Mutants Representing Human Complementation Group III Kanji Okumoto, Akemi Bogaki,* Keita Tateishi, Toshiro Tsukamoto,* Takashi Osumi,* Nobuyuki Shimozawa,† Yasuyuki Suzuki,† Tadao Orii,†,1 and Yukio Fujiki2 Department of Biology, Kyushu University Faculty of Science, Fukuoka 812-81, Japan; *Department of Life Science, Himeji Institute of Technology, Kamigori, Hyogo 678-12, Japan; and †Department of Pediatrics, Gifu University, Gifu 500, Japan
tions in essential metabolic pathways, including b-oxidation of very long fatty acid and synthesis of etherglycerolipids such as plasmalogen [1]. Human peroxisomedeficient disorders are autosomal recessive [2] and include at least 10 different known genotypes [3–7], with three distinct phenotypes, manifested in cerebrohepatorenal Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease [1, 2]. To investigate mechanisms involved in peroxisome biogenesis and the primary defects of human peroxisome assembly disorders, we earlier isolated three mutually distinct peroxisome-deficient Chinese hamster ovary (CHO)3 cell mutants, Z24, Z65, and ZP92, by colony-autoradiographic screening and the 9-(1*-pyrene)nonanol/ultraviolet (P9OH/UV) selection methods [6, 8]. The mutant cells resembled fibroblasts from patients with peroxisome-deficient disease such as Zellweger syndrome, with defects in peroxisome assembly, but the synthesis of peroxisomal proteins was normal. Complementation groups between three CHO mutants and nine groups of fibroblasts from patients with peroxisome-deficient disorders were defined [6, 9]. Peroxisome assembly factor-1 (PAF-1, termed Pex2p by a unified nomenclature [10]), a 35-kDa peroxisomal integral membrane protein, proved to be essential for restoration of peroxisome biogenesis in the CHO mutant Z65 [11]. The PEX2 gene was shown to be the first pathogenic gene of Zellweger syndrome; a homozygous nonsense mutation (Arg119Stop) was the genetic cause of this syndrome in a complementation group F patient in Japan (the same as X in the United States and 5 in
We made use of the 9-(1*-pyrene)nonanol/ultraviolet (P9OH/UV) method and isolated peroxisome-deficient mutant cells. TKa cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1) were used to avoid frequent isolation of the Z65-type, PEX2-defective mutants. P9OH/UV-resistant cell colonies were examined for the intracellular location of catalase, a peroxisomal matrix enzyme, by immunofluorescence microscopy and using anti-catalase antibody. As six mutant cell clones showed cytosolic catalase, there was likely to be a deficiency in peroxisome assembly. These mutants also showed the typical peroxisome assembly-defective phenotype, including significant decrease of dihydroxyacetonephosphate acyltransferase, the first step key enzyme in plasmalogen synthesis, and loss of resistance to 12-(1*-pyrene)dodecanoic acid/UV treatment. By transfection of Pex2p and Pex6p (formerly PAF-2) cDNAs and cell fusion analysis between the CHO cell mutants, two mutants, ZP104 and ZP109, were found to belong to a novel complementation group. Further complementation analysis using fibroblasts from patients with peroxisome biogenesis disorders revealed that the mutants belonged to human complementation group III. Taken together, ZP104 and ZP109 are in a newly identified fifth complementation group in CHO mutants reported to date and represent the human complementation group III. q 1997 Academic Press
INTRODUCTION
Peroxisome is a single membrane-bounded, ubiquitous intracellular organelle in eukaryotes and func-
3
Abbreviations used: AOx, acyl-CoA oxidase; CHO, Chinese hamster ovary; DHAP-ATase, dihydroxyacetonephosphate acyltransferase; FCS, fetal calf serum; Hygr, resistant to hygromycin; PMP70, 70-kDa peroxisomal integral membrane protein; Ouar, resistant to ouabain; P9OH/UV, 9-(1*-pyrene) nonanol/ultraviolet; P12, 12-(1*pyrene)dodecanoicacid; PAF-1 and PAF-2, peroxisome assembly factor-1 and -2, respectively; PTS1R, peroxisome targeting signal type 1 receptor; TGr, resistant to 6-thioguanine.
1
Present address: Chubu Women’s College, Seki, Japan. To whom correspondence and reprint requests should be addressed at Department of Biology, Kyushu University Faculty of Science, 6-10-1 Hakozaki, Fukuoka 812-81, Japan. Fax: (092) 6422645. E-mail: [email protected]. 2
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FIG. 1. Immunofluorescent staining of wild-type CHO cells, mutants, and hybrid cells. (A) Cells were incubated with rabbit anti-rat catalase antibody. (a) Wild-type CHO cells; (b) PEX2-transformed wild-type, TKa; (c–h) mutants ZP104, ZP105, ZP106, ZP107, ZP108, and ZP109, respectively. Scale, 30 mm. (B) Cells were stained with anti-rat PMP70 antibody. (a) TKa; (b) ZP104; (c) ZP109. Scale, 20 mm.
Europe) [9, 12]. Recently, peroxisome assembly factor-2 (PAF-2) cDNA, termed PEX6, that restored peroxisome assembly in the mutant ZP92 as well as the fibroblasts derived from patients with Zellweger syndrome of complementation group C in Japan (the same as group IV in the United States and group 3 in Europe) was cloned [13, 14] and was found to encode a member of the putative ATPase (AAA) family [15]. Several other complementing genes of yeast mutants such as Saccharomyces cerevisiae PAS1 (PEX1), PEX6 from Pichia pastoris (formerly PAS5), and Yallowia lipolytica (formerly PAY4) also belong to this family [16–18]. Thus, peroxisome assembly-defective CHO cell mutants are indeed a useful system for the study of peroxisome biogenesis
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and for elucidating primary defects of human peroxisome disorders. We report here a new complementation group of CHO cell mutants that apparently belongs to the human complementation group III. MATERIALS AND METHODS Selection of peroxisome-deficient CHO cell mutants. Wild-type CHO-K1 cells were cultured in Ham’s F12 medium supplemented with 10% fetal calf serum (FCS) and transfected with rat Pex2p cDNA, a plasmid pcD65K [11], as described. Stable transformant, TKa, selected with G418, was found to express rat PEX2 mRNA [19]. TKa cells were treated with N-methyl-N*-nitro-N-nitrosoguanidine (Nacalai Tesque, Kyoto, Japan) and grown in F12 medium containing
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FIG. 2. Complementation analysis by cDNA transfection. CHO cell mutants ZP106 and ZP109 were each transfected with cDNA coding for Pex2p or Pex6p (formerly PAF-1 and PAF-2, respectively) and stained with anti-catalase antibody. (a and b) ZP106; (c and d) ZP109. Peroxisomes were noted only in b. Scale, 30 mm. 10% FCS for 24 h. The growth medium was changed to the F12containing 10% FCS and 6 mM P9OH (Molecular Probes, Inc., Eugene, OR) and the cells were further cultured for 20 h. The cells were washed once with F12 and incubated for 6 h in F12 with 10% FCS. After a thorough wash of the cells with F12, mutants deficient in peroxisomes were selected by a 2-min exposure to UV light, as described [6]. Assays. Dihydroxyacetonephosphate acyltransferase (DHAPATase) [20], the latency of catalase [6], and resistance to P9OH/ UV and 12-(1*-pyrene)dodecanoic acid (P12)/UV treatments [6] were measured, as described. P12 was from Molecular Probes, Inc. Morphological analysis. Peroxisomes in CHO cells were visualized by indirect immunofluorescence light microscopy using rabbit antibody to rat liver catalase and those in human fibroblasts were detected with rabbit anti-human catalase antibody, as described [6]. Antigen–antibody complex was detected by fluorescein isothiocyanate-labeled goat anti-rabbit immunogloblin G antibody (Cappel, Durham, NC; ZYMED Laboratories, Inc., South San Fransisco, CA) under a Carl Zeiss Axioskop FL microscope (Oberkochen, Germany). Several CHO mutant cell clones were stained with rabbit antibody to 70-kDa peroxisomal integral membrane protein (PMP70) [6]. DNA transfection. DNA transfection to CHO mutant cells was done with plasmids pcD65K and pUcD2r92A, mammalian expression vectors containing cDNAs for rat Pex2p and Pex6p, respectively [11, 13]. Two methods were used: one was electroporation [13], except that cells suspended in phosphate-buffered saline (magnesium- and
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calcium-free) at 1 1 106 cells/ml were mixed with 10 mg plasmid DNA and electroporated using an electroporator, Model ECM600 (BTX Inc., San Diego, CA) on a setting of 300 V, 400 mF, and 72 V; the other method was liposome-mediated transfection [21] but with some modification. Briefly, cells that had been plated on a coverslip 1 day before transfection at 8 1 104 cells in a 35-mm dish were washed twice with Hepes-buffered saline (20 mM Hepes–KOH, 150 mM NaCl, pH7.4) supplemented with 1 mM CaCl2 and 0.5 mM MgCl2 . Three micrograms of plasmid DNA and 10 mg of cationic liposome (O,O*-ditetradecanolyl-N-(trimethylammonioacetyl) diethanolamine chloride; 14Dea2, a gift from A. Ito) prepared by sonication were separately diluted with 300 ml of Hepes-buffered saline containing CaCl2 and MgCl2 . Both solutions were mixed and poured over the cells. After incubation for 1 h at 377C, the DNA–liposome mixture was removed from the culture medium by aspiration and fresh F12 medium with 10% FCS was added. The cells were cultured for 2 days and then were further incubated overnight in 2 ml of serum-free F12 medium before immunostaining. Cell fusion and labeling of cell protein. We used variants of cell mutants resistant to 6-thioguanine (TGr) plus ouabain (TGrOuar) [8]. CHO cells resistant to 6-thioguanine and hygromycin (TGrHygr) were isolated by transfection of a plasmid pMiw hph (a gift from H. Kondo) to TGr variants, using the calcium phosphate method [22] followed by selection in the presence of 200 U/ml hygromycin B (Wako, Osaka, Japan). Parent CHO cells and the CHO cells to be examined were cocultured for 24 h and then fused with the use of polyethylene glycol,
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TABLE 1 Complementation Group Analysis of CHO Cell Mutants by cDNA Transfection Transfection of cDNA coding for CHO mutant ZP104 ZP105 ZP106 ZP107 ZP108 ZP109 Z65 ZP92
Pex2p
Pex6p
0 0 0 0 0 0 / 0
0 0 / 0 0 0 0 /
Note. Cells were stained with anti-rat catalase antibody 3 days after cDNA transfection. (/) Peroxisomes were complemented; (0) not complemented.
as described [6], except that selection of fused cells was carried out with 1 mM ouabain or 200 U/ml hygromycin B (Wako, Osaka, Japan). Cell fusion of CHO mutant TGr variants with human fibroblasts was done as described [6]. Metabolic labeling of cells with 20 mCi/ml of [35S]methionine and [35S]cysteine (New England Nuclear, Boston, MA) for 24 h in F12 medium and immunoprecipitation of peroxisomal proteins from cell lysates were done, as described [8].
RESULTS
Isolation and Morphological Analysis of CHO Cell Mutants PEX2-transformed CHO-K1, TKa cells were mutagenized and those cells resistant to P9OH/UV treatment were selected. Viable cell colonies were examined for peroxisome morphology, using an anti-catalase antibody. Six mutant cell clones, ZP104, ZP105, ZP106,
ZP107, ZP108, and ZP109, were isolated by cell cloning from mutant colonies with diffuse catalase present in the cytoplasm, with clear presence in the cytosol, and a phenotype similar to that previously isolated mutants, Z24, Z65, and ZP92 [6, 8] (Fig. 1A, c–h). In most P9OH/ UV-resistant cells as well as the wild-type CHO-K1 and TKa, catalase was detected in numerous particles, presumably peroxisomes (Fig. 1A, a and b). At present, we have no explanation as to why there was occasional resistance of some peroxisome-positive cells to the P9OH/UV treatment. Mutants ZP104 and ZP109 were then stained with antiserum against PMP70 (Fig. 1B, b and c). Larger but fewer particles immunoreactive with anti-70 IMP antibody were detected, consistent with findings in previously isolated CHO cell mutants [6] and similar to ‘‘peroxisomal ghost’’ vesicles in the fibroblasts from Zellweger patients [23–25]. These particles were also noted in other mutants, ZP105 to ZP108 (not shown). There was a punctate staining pattern in the wild-type cells, as seen with anti-catalase antibody (Fig. 1B, a). Complementation Group Analysis by cDNA Transfection Mutant cells, ZP104 to ZP109, were each transfected with cDNA for Pex2p or Pex6p. Transient transfectants were analyzed by indirect immunofluorescence microscopy using anti-catalase antibody. Numerous peroxisomes were noted only in ZP106 transfected with Pex6p cDNA, thereby indicating that ZP106 belongs to the complementation group of ZP92, whereas all of the other transfectants showed cytosolic catalase (Fig. 2 and Table 1). As a control, peroxisome assembly in Z65 cells was restored by Pex2p cDNA, but not with Pex6p cDNA, consistent with earlier findings [11]. Conversely, peroxisome formation in ZP92 cells was com-
FIG. 3. Cell fusion analysis of CHO cell mutants. CHO cell mutants ZP104 and ZP105 were fused with ZP109 and stained with antirat catalase antibody. (a) Fusion of ZP104 with ZP109; (b) ZP105 with ZP109. Scale, 20 mm.
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TABLE 2 Complementation Group Analysis of CHO Mutants by Cell Fusion
ZP104 ZP105 ZP107 ZP108 ZP109
ZP104
ZP105
ZP107
ZP108
ZP109
N.D. / / / 0
/ N.D. / / /
/ / N.D. 0 /
/ / 0 N.D. /
0 / / / 0
fused cells of each mutant with the wild-type CHO-K1 showed catalase-positive particles as numerous as in the wild type, suggesting that a lesion(s) of the allele(s) in the mutants are recessive (Table 3). The mutant ZP109 was then used for further analyses. Latency of Catalase
plemented only with Pex6p and not by Pex2p expression [13]. Accordingly, the CHO mutants isolated in this study, except for ZP106, belong to a complementation group(s) different from those of Z65 and ZP92. Moreover, no Z65-type mutant was isolated, thereby clearly demonstrating the efficacy of TKa cells in isolating mutants devoid of PEX2 deficiency.
The cellular location of catalase was also examined by the digitonin assay [6, 8]. At 100 mg/ml of digitonin, full activity of catalase was detected in the mutant ZP109 as in Z65, whereas Ç60% of the activity was latent in the wild-type cells, CHO-K1 and TKa (Fig. 5). Mutants ZP104, ZP105, ZP106, ZP107, and ZP108 also showed catalase latency similar to that of ZP109 (data not shown). Treatment with 300 mg/ml of digitonin released full catalase activity of the wild type. These results were interpreted to mean that catalase is localized in the cytosol in these mutants. Cell hybrids of ZP109 and Z65 showed the same profile of catalase latency as the parent TKa and CHO-K1, strongly suggesting that these mutants differ in the complementation group, consistent with observations in the cell fusion study and the morphological analysis.
Cell Fusion Analysis
Cell Fusion with Fibroblasts from Zellweger Patients
To classify six CHO mutants, ZP104 to ZP109, all combinations of cell fusion excluding ZP106 were set up (Fig. 3, Table 2). Fusion of ZP104 with ZP109 and ZP107 with ZP108 produced no peroxisomal punctate structure, indicating that the respective pair of mutants belong to the same complementation group (Fig. 3a, Table 2). Peroxisomes were noted in hybrid cells between ZP105 and ZP109, which means that ZP105 is distinct from ZP109 (Fig. 3b). Likewise, peroxisomes were complemented in all of the other fused cells, except for a homologous hybrid of ZP109 (Table 2). Accordingly, the mutants isolated in this study could be classified into four complementation groups, i.e., ZP104 and ZP109 are in one group; ZP105, ZP106, and ZP107, as well as ZP108 are, respectively, in three different groups. Next, the CHO mutants were fused with previously isolated Z24 and ZP102 (the same complementation group as human group II in the United States [19]). Peroxisomes were not complemented in assembly by cell fusion between ZP105 and ZP102, ZP107 and Z24, and ZP108 and Z24, while other cell hybrids such as those of ZP109 with Z65, ZP92, Z24, and ZP102 did have peroxisome particles (Fig. 4, Table 3). These observations strongly suggest that ZP104 and ZP109 belong to a novel complementation group of CHO mutants and that ZP107 and ZP108 are in the same complementation group as Z24. ZP105 belongs to complementation group ZP102, the same as human group II. Moreover,
To determine to which human complementation group the CHO mutant ZP109 belonged, cell fusion was carried out between ZP109 and human fibroblasts from peroxisome-deficient patients with Zellweger syndrome; complementing genes were not delineated. Peroxisomes were not complemented in fused cells of ZP109 and fibroblasts from a Zellweger patient of complementation group III, whereas numerous punctate catalase-containing structures, peroxisomes, were noted in the hybrid cells of ZP109 and fibroblasts from a patient of complementation group B in Japan (the same as group VII in the United States) (Fig. 6). Like-
Note. Cell fusion was done as under Materials and Methods. Unselected fused cells were stained with anti-rat catalase antibody. (/) Peroxisomes were complemented; (0) not complemented; N.D., not done.
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TABLE 3 Complementation Group Analysis of CHO Mutants by Cell Fusion
ZP104 ZP105 ZP107 ZP108 ZP109
Z65a
ZP92a
Z24a
ZP102b
CHO-K1a
N.D. N.D. N.D. N.D. /
N.D. N.D. N.D. N.D. /
/ / 0 0 /
/ 0 / / /
/ / / / /
Note. Cell fusion was done as for Table 2. Fused cells were selected and stained with anti-rat catalase antibody. (/) Peroxisomes were complemented; (0) not complemented; N.D., not done. a,b Cells resistant to 6-thioguanine plus ouabain [6, 8] and 6-thioguanine plus hygromycin, respectively.
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FIG. 4. Complementation group analysis of CHO cell mutants. Mutants were pairwise fused and after selection, the hybrids were stained with anti-catalase antibody. (a–c) Fused cells of ZP105 with ZP102TGrHygr, ZP107 with Z24TGrOuar, and ZP108 with Z24TGrOuar, respectively; (d–h) hybrid cells of ZP109 with Z65TGrOuar, ZP92TGrOuar, Z24TGrOuar, ZP102TGrHygr, and the wild-type CHO-K1TGrOuar, respectively. Scale, 20 mm.
wise, peroxisomes were noted in fused cells between ZP109 and peroxisome-deficient patient fibroblasts of groups A (the same as group VIII in the United States), D (the same as group IX), G [7], and VI, respectively (Table 4). Taken together, ZP109 apparently belongs to human complementation group III, distinct from groups A, B, D, G, and VI. Properties of CHO Cell Mutants Mutants ZP105 and ZP109 were severely defective in DHAP-ATase activity and were sensitive to P12/UV
FIG. 5. Latency of catalase in wild-type, mutant, and hybrid cells. Latency of catalase was determined as described [8]. (s) Wildtype CHO-K1; (n), CHO-TKa; (l), ZP109; (m), Z65; (h), ZP109 1 Z65TGrOuar. The results were represented as averages of duplicate assays.
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treatment as was noted for mutants Z24, Z65, and ZP92 [6, 8], while the DHAP-ATase level in TKa cells was as high as that of the wild-type CHO-K1 (Table 5). DHAP-ATase activity of other mutants, ZP104, ZP106, and ZP107, was also low (not shown). After cell culture in the presence of P9OH followed by a short exposure to UV, over 90% of ZP105 and ZP109 cells survived, but only less than 0.001% of TKa cells were viable, in good agreement with the previous findings for the wildtype CHO-K1 and mutants Z24, Z65, and ZP92 [6, 8]. The mutant cells were highly sensitive to P12/UV treatment, but nearly 70% of TKa cells were resistant, again consistent with previous deductions [8]. These cell-phenotypic properties were also noted for the other mutants, ZP104, ZP106, ZP107, and ZP108 (not shown). Biogenesis of peroxisomal enzymes was investigated in the mutant ZP109 and its hybrid with Z65 that had been labeled with [35S]methionine and [35S]cysteine. Acyl-CoA oxidase (AOx), the first enzyme of the peroxisomal b-oxidation system, is a heterodimer comprising 75-kDa A, 53-kDa B, and 22-kDa C polypeptide components [8, 26]; B and C are derived from A [27]. All three 35 S-labeled polypeptide components were evident in the wild-type CHO-K1 and TKa (Fig. 7, lanes 1 and 2) with a faint band of C attributed to the low content of methionine and cysteine [8, 26], whereas the reduced 75-kDa 35 S-labeled A form of AOx, but not converted forms (53-kDa B and 22-kDa C components), was detected in ZP109 and Z65 because of the rapid degradation of AOx-A (Fig. 7, lanes 3 and 4), as noted in the mutants Z24 and ZP92 [6, 8]. The third enzyme of the peroxi-
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FIG. 6. Cell fusion of ZP109 with fibroblasts from Zellweger patients. (a and b) Cell hybrids of ZP109 with fibroblasts from Zellweger patients of human complementation groups III and B, respectively. Note the numerous peroxisomes in b but not in a. Scale, 20 mm.
somal b-oxidation system, 3-ketoacyl-CoA thiolase (thiolase), is synthesized as a larger precursor of 44 kDa and then processed to a 41-kDa mature form [8]. Only a 44-kDa 35S-labeled precursor of thiolase was detectable in ZP109 and Z65, consistent with the finding in Z24 and ZP92 [6, 8], although the 41-kDa mature protein was apparent in the wild-type cells, CHO-K1 and TKa (Fig. 7, lanes 6–9). Thus, peroxisomal proteins are apparently synthesized at a normal level in mutants, as concluded from previous studies [6, 8]. In the hybrid of ZP109 with Z65, appropriate processing of AOx and thiolase was evident, as in the wild type (Fig. 7, lanes 5 and 10, respectively). These observations support the notion that the CHO mutant ZP109 belongs to a complementation group distinct from Z65. TABLE 4 Complementation Analysis of ZP109 by Cell Fusion with Fibroblasts from Patients with Peroxisome-Deficient Disorders Complementation groups of patient fibroblastsa
ZP109b
A
B
D
G
III
VI
/
/
/
/
0
/
Note. After cell fusion, fused cells were selected and stained with anti-human catalase antibody as described [6]. (/) Peroxisomes were complemented; (0) not complemented. a Used fibroblasts were from peroxisome-deficient patients belonging to complementation groups whose complementing genes were not identified. b ZP109 cells resistant to 6-thioguanine.
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DISCUSSION
A search for genes essential for peroxisome assembly has entailed significant efforts, as has elucidation of primary defects of peroxisome biogenesis disorders, including Zellweger syndrome and neonatal adrenoleukodystorophy. cDNAs coding for Pex2p and Pex6p were cloned by phenotypic complementation of CHO mutants Z65 and ZP92, respectively [11, 13]. Mutations in the PEX2 gene proved to be the cause of Zellweger syndrome of complementation group F (the same as groups X and 5) [9] (Table 6). Human cDNA encoding a receptor, PEX5, for peroxisome targeting signal type 1, SKL motif was independently isolated by an expressed sequence tag search with P. pastoris PAS8 [28], immunoscreening of a cDNA expression library [29], and a yeast two-hybrid system [30]. PEX5 and PEX6 were shown to be responsible for primary defects in the patients of complementation groups II and C (the same as group IV) disorders [13, 14, 28, 29, 31]. In the present work, we investigated by mutant selection peroxisome-deficient CHO cell mutants distinct from the previous Z24, Z65, and ZP92. By making a modification with use of the TKa, the wild-type expressing rat Pex2p mRNA [19], frequent isolation of Z65type mutants [6] was avoided. After several cycles of the mutant isolation procedure, i.e., P9OH/UV treatment followed by indirect immunofluorescence microscopy, six mutant cell clones were isolated, from which two, ZP104 and ZP109, were found to belong to a newly defined complementation group. All of the CHO cell
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TABLE 5 Characteristics of Peroxisome-Deficient CHO Cell Mutants CHO cell
Peroxisomes
Catalase latency (%)
DHAP-ATase (%)
P9OH/UV (%)
P12/UV (%)
Wild type TKa ZP105 ZP109
/ / 0 0
63 63 0 0
100 100 11 10
õ0.001a õ0.001 93 99
87a 66 õ0.001 õ0.001
Note. Catalase latency represents peroxisomal catalase, calculated as described [8]. Peroxisomal DHAP-ATase was measured as described [20]. Survival rates of P9OH/UV- and P12/UV-resistant cells were expressed as percentages of the unselected control [6]. a From reference [6].
mutants we isolated herein showed typical, common properties characterized for previously isolated Z24, Z65, and ZP92, including a recessive lesion(s), lacking morphologically recognizable peroxisomes, no latency of catalase, a severe loss of DHAP-ATase, and high sensitivity to the P12/UV treatment but with a normal synthesis of peroxisomal proteins. The mutants also contained peroxisomal ghost-like vesicular structures, as described for all of the CHO mutants [6] and for fibroblasts from patients with peroxisome-deficient dis-
FIG. 7. Biogenesis of peroxisomal proteins. Cells were labeled for 24 h with [35S]methionine and [35S]cysteine. Cell types are indicated at the top. Immunoprecipitation was done with rabbit antirat acyl-CoA oxidase (AOx) and 3-ketoacyl-CoA thiolase antibodies. Immunoprepicitates were analyzed by SDS–PAGE (12% polyacrylamide gel). Radioactive polypeptide bands were detected using a FujiX BAS1000 Bio-Imaging Analyzer (Fuji Photo Film, Tokyo, Japan). Exposure 20 h. Arrows show the position of AOx components; open and solid arrowheads indicate a larger precursor and mature protein of thiolase, respectively.
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ease [23–25]; however, the significance of the ‘‘peroxisomal ghost’’ is not clear. Accordingly, mutants isolated in this study represent typical somatic mammalian cell mutants defective in peroxisome biogenesis. By complementation analysis, six mutants were grouped into four (Table 6). Mutant ZP105 is in the same complementation group as ZP102 which belongs to human complementation group II [19], where the PTS1 receptor is responsible for the primary defect [28, 29]. Thus, ZP105 may serve as a model mammalian cell system in which to examine functions and dysfunctions of PTS1 receptor at molecular and cellular levels. ZP106, like ZP92, is in the human complementation group C (the same as groups IV and 3) in which malfunction of Pex6p is evident [13]. ZP107 and ZP108 belong to the complementation group Z24, human complementation group E (the same as groups 1 and 2). ZP104 and ZP109 were classified into the complementation group III. Accordingly, mutants ZP104 and ZP109 are in a newly identified, fifth complementation group of CHO cell mutant clones that belong to complementation group III, among 10 human complementation groups [3–7]. The import of both PTS1- and PTS2 proteins is defective in ZP109 (not shown), consistent with the results using fibroblasts of complementation group III patients [32]. Therefore, mutants ZP104/ ZP109 and ZP107/ZP108 will be useful for cloning genes essential for peroxisome biogenesis in complementation groups III and I (the same as groups E and 2) as well as for studies on mechanisms of peroxisome assembly in mammals. At this writing, only three responsible genes, PEX2 [9, 11, 33, 34], PEX5 [28, 29], and PEX6 [13, 14, 31] are elucidated. During the course of this study, P9OH/UV-resistant, viable cell colonies but with the peroxisomal punctate catalase-staining pattern were evident. Similar findings were reported for murine macrophage-like cell lines isolated using the same method [35]. Several explanations such as reduced incorporation of P9OH by mutation(s) can be given for the viability of peroxisome-
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TABLE 6 Complementation Groups and Complementing Genes of Peroxisome Deficiency Human fibroblasts Japan (Gifu)
U.S.A. (Kennedy)
A B C D E F
VIII VII IV IX I X II III VI
Europe (Amsterdam)
3 2 5 4
Gd
Phenotype ZS, NALD, IRD ZS, NALD ZS ZS ZS, NALD, IRD ZS ZS, NALD ZS NALD ZS
CHO mutants
ZP106, ZP92b
Complementing gene
PEX6 (PAF-2)
a
Z24, ZP107, ZP108 Z65a ZP102,c ZP105 ZP104, ZP109
PEX2 (PAF-1) PEX5
Note. ZS, Zellweger syndrome; NALD, neonatal adrenoleukodystrophy; IRD, infantile Refsum disease. a,b From refs. [6, 8]; c,dfrom [19, 7].
Cregg, J. M., Dodt, G., Fujiki, Y., Goodman, J. M., Just, W. W., Kiel, J. A. K. W., Kunau, W.-H., Lazarow, P. B., Mannaerts, G. P., Moser, H., Osumi, T., Rachubinski, R. A., Roscher, A., Subramani, S., Tabak, H. F., Tsukamoto, T., Valle, D., van der Klei, I., van Veldhoven, P. P., and Veenhuis, M. (1996) J. Cell Biol. 135, 1–3.
positive cells under the conditions used; therefore, further investigation is warranted. We thank H. W. Moser for patients’ fibroblasts of complementation groups III and VI, A. Ito for supply of liposomes, H. Kondo for the generous gift of a plasmid, K. Ohashi for technical advice, and K. Mizuno and M. Ohara for helpful comments. This work was supported in part by Grants-in-Aid for Scientific Research (07408016 to Y.F.) and by grants (to Y.F.) from Uehara Memorial Foundation, Mitsubishi Foundation, Nagase Science and Technology Foundation, Naito Foundation, and Ciba-Geigy Foundation (Japan) for the Promotion of Science.
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