J. Biochem. I l l , 783-787 (1992)
^-Oxidation of Butyrate, the Short-Chain-Length Fatty Acid, Occurs in Peroxisomes in the Yeast Candida tropicalis Tatsuo Kurihara,* Mitsuyoshi Ueda,* Hirofumi Okada,* Naomi Kamasawa," Nobuko Naito," Masako Osumi,** and Atsuo Tanaka' 1 'Laboratory of Industrial Biochemistry, Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-01; and "Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Mejiro-dai, Bunkyo-ku, Tokyo 112 Received for publication, January 21, 1992
When an n-alkane-utilizable yeast, Candida tropicalis pK233, was cultivated on butyrate, the fatty acid of shortest chain-length for /9-oxidation, as the sole source of carbon and energy, catalase and the enzymes of the fatty acid /9-oxidation system were inducibly synthesized at high levels. As in the alkane-grown cells, the proliferation of peroxisomes was harmonized with the induction of peroxisomal enzymes. The results of subcellular fractionation and immunoelectronmicroscopy indicated the localization of these enzymes in peroxisomes, not in mitochondria. It was suggested that only peroxisomes have a role in fatty acid /9-oxidation in the yeast cells, unlike in mammalian cells, in which cooperation between peroxisomes and mitochondria is essential.
Many organisms can utilize fatty acids as the source of carbon and energy. During assimilation, fatty acids are activated to fatty acyl-CoAs and degraded to acetyl-CoA via a /9-oxidation pathway. In eukaryotic cells, this pathway is localized either in mitochondria and/or in peroxisomes (1-4). In mammalian cells, long-chain substrates are supposed to be degraded in peroxisomes and mediumor short-chain substrates in mitochondria (5). When microorganisms are cultivated on long-chain fatty acids or n-alkanes to be oxidized to fatty acids, the activity level of the /9-oxidation system increases; and the /J-oxidation system is detected only in peroxisomes (4, 6-9). However, the (3- oxidation system in eukaryotic microorganisms grown on short-chain fatty acids has not been studied in detail. Candida tropicalis is an invaluable microorganism in the study of fatty acid metabolism and biogenesis of peroxisomes. This yeast can utilize various carbon sources, such as glucose, acetate, propionate, and n-alkanes (10, 11). When it is cultivated on n-alkanes (CiO-Cu), the enzymes of the fatty acid /9-oxidation system are inducibly synthesized and specifically localized in peroxisomes (4, 6), which proliferate remarkably under these conditions (10). Fatty acids derived from n-alkanes are degraded via the peroxisomal /9-oxidation system. In the cells grown on glucose or acetate, induction of the enzymes of the /9-oxidation system and proliferation of peroxisomes were not observed (6, JO). In the propionate-grown cells, the activity levels of the enzymes of the /9-oxidation system increase above those in the glucose-grown cells and the volume of peroxisomes increases, though the physiological role of these enzymes in the propionate-grown cells is obscure (21, 12). In the present study, we have tried to cultivate this yeast on butyrate, which is the fatty acid of shortest chain-length to be degraded via the /9-oxidation system. We also examined 1
To whom correspondence should be addressed.
Vol. I l l , No. 6, 1992
the degree of peroxisome development, the activity level of the /9-oxidation enzymes, and their subcellular localization. Based on the results obtained, we discuss the difference in peroxisomal function in fatty acid /9-oxidation for shortchain-length substrates between yeast cells and mammalian cells. MATERIALS AND METHODS Cultivation of Yeast— C. tropicalis pK233 (ATCC 20336) was cultivated aerobically at 30'C in a medium containing glucose (16.5 g/liter), sodium acetate-3H2O (13.6g/liter), sodium propionate (10.0 g/liter), sodium butyrate (11.0 g/liter), or n-alkane mixture (CiO-Cu) (10 ml/liter) as the sole source of carbon and energy. The basic composition of the medium was described previously (11). In this study, 0.5 ml/liter of Tween 80 was added to each medium and the initial pH was adjusted to 6.0. The cultivation was carried out for 8 h on glucose and acetate, 22 h on propionate, 36 h on butyrate, and 17 h on alkanes. Preparation of Cell-Free Extracts and Subcellular Fractionation—Cell-free extracts were prepared by disintegrating the cells by sonication in 50 mM potassium phosphate buffer (pH7.2) containing 10% glycerol (11). Subcellular fractionation of the butyrate-grown cells was carried out in a similar manner to that of the propionategrown cells (12) except for the sucrose concentration used for density gradient centrifugation. Enzyme and Protein Assay—The activities of the enzymes of the fatty acid /9-oxidation system (acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and thiolase) were measured as described previously (6, 13). Thiolase was assayed using either acetoacetyl-CoA (for acetoacetyl-CoA thiolase activity) or 3-ketooctanoyl-CoA (for 3-ketoacyl-CoA thiolase activity) (14, 15). Catalase, cytochrome oxidase, and proteins were assayed by the methods described (16).
783
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M N
CM
B «vCW
J. Biochem.
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Peroxisomal fi-OxidaUon System in Butyrate-Grown C. tropicalis Electronmicroscopy—For electronmicroscopy, the butyrate-grown and glucose-grown cells were prefixed with 1% glutaraldehyde, and post-fixed with osmium tetraoxide as described previously {17). Immunoelectronmicroscopy of the butyrate-grown and alkane-grown cells was carried out as described (18, 19) using anti-(3-ketoacyl-CoA thiolase) antiserum. This antiserum was prepared using the enzyme purified from particulate fractions (20,000 Xg) containing peroxisomes and mitochondria (15). Chemicals—3-Ketooctanoyl-CoA was prepared enzymatically from octenoyl-C!oA (20), synthesized by the mixed anhydride method (21). Other chemicals were obtained from commercial sources. RESULTS Enzyme Activities in the Cells Grown on Various Carbon Sources— C. tropicalis was found to grow on butyrate as the sole source of carbon and energy, when the cultivation
Propionate Butyrate
conditions were optimized as described in "MATERIALS AND METHODS." The activities of catalase and the enzymes of the fatty acid /?-oxddation system in the cell-free extracts were compared among the cells grown on various carbon sources. As shown in Fig. 1, the activities of the enzymes of the fatty acid p- oxidation system were higher in the butyrate-grown and alkane-grown cells than in the glucosegrown, acetate-grown, and propionate-grown cells. The activities of enoyl-CoA hydratase, acetoacetyl -CoA thiolase, and 3-ketoacyl-CoA thiolase in the butyrate-grown cells were equal to or higher than those in the alkane-grown cells. The activity of acyl-CoA oxidase in the butyrategrown cells was lower than that in the alkane-grown cells, but it was higher than that in the cells grown on other carbon sources. The level of catalase activity in the butyrate-grown cells was comparable to that in the propionate-grown cells, but higher than that of the glucose- and acetate-grown cells. Electronmicroscopical Observation—The increase in the activity of catalase suggested the proliferation of peroxisomes in the butyrate-grown cells. This was confirmed by electronmicroscopy, as shown in Fig. 2, which revealed peroxisomes to be present profusely in the butyrate-grown cells but rarely in the glucose-grown cells. Subcellular Localization of Catalase and the Enzymes of the Fatty Acid ^-Oxidation System—As described above, catalase and the enzymes of the fatty acid /?- oxidation
Acjrl-CoA oxidase
Glucose
Butyrate
400 Enoyl-CoA hydntase
3-Ketoacyl-CoA
600
thiolase
(«!...„-'.
o Cr
30 1
15 | |
15
0 30
S--
20 •
Butyrate
3 0
100 200 300 Acetoacetyl-CoA thiolase (naol.nln"'.»g~')
Fig. 1. Specific activities of catalase and the enzymes of the fatty acid ^-oxidation system in C tropicalis cells grown on different carbon sources. (A) Catalase; (B) acyl-CoA oxidase; (C) enoyl-CoA hydratase; (D) 3-ketoacyl-CoA thiolase; (E) acetoacetylCoA thiolase. The profile of 3-hydroxyacyl-CoA dehydrogenase activity was the same as that of enoyl-CoA hydratase on the same multi-functional protein (28). Fig. 2. Electronmicrographs of the butyrate-grown (A) and glucose-grown cells (B) of C tropicalis. CM, cell membrane; CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. Bar, Vol. I l l , No. 6, 1992
20 • J0 ' 40 "41.3W2.5I 50 Sucrose concentration
(\)
20 ' 30 I *0 M l . 3 « 2 . S l Sucrose concentration
50 I (\)
Fig. 3. Particulate localization of emymes in the P, fraction (20,0OOXff pellet). The volume of each fraction was as follows: 1, 3.75 ml; 2-5, 2.5 ml each; 6, 1.25 ml. (A) Protein; (B) cytochrome oxidase; (C) catalase; (D) acyl-CoA oxidase; (E) enoyl-CoA hydratase; (F) 3-ketoacyl-CoA thiolase; (G) acetoacetyl-CoA thiolase.
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^-Oxidation of Butyrate, the Short-Chain-Length Fatty Acid, Occurs in Peroxisomes in the Yeast Candida tropicalis Tatsuo Kurihara,* Mitsuyoshi Ueda,* Hirofumi Okada,* Naomi Kamasawa," Nobuko Naito," Masako Osumi,** and Atsuo Tanaka' 1 'Laboratory of Industrial Biochemistry, Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-01; and "Department of Chemical and Biological Sciences, Faculty of Science, Japan Women's University, Mejiro-dai, Bunkyo-ku, Tokyo 112 Received for publication, January 21, 1992
When an n-alkane-utilizable yeast, Candida tropicalis pK233, was cultivated on butyrate, the fatty acid of shortest chain-length for /9-oxidation, as the sole source of carbon and energy, catalase and the enzymes of the fatty acid /9-oxidation system were inducibly synthesized at high levels. As in the alkane-grown cells, the proliferation of peroxisomes was harmonized with the induction of peroxisomal enzymes. The results of subcellular fractionation and immunoelectronmicroscopy indicated the localization of these enzymes in peroxisomes, not in mitochondria. It was suggested that only peroxisomes have a role in fatty acid /9-oxidation in the yeast cells, unlike in mammalian cells, in which cooperation between peroxisomes and mitochondria is essential.
Many organisms can utilize fatty acids as the source of carbon and energy. During assimilation, fatty acids are activated to fatty acyl-CoAs and degraded to acetyl-CoA via a /9-oxidation pathway. In eukaryotic cells, this pathway is localized either in mitochondria and/or in peroxisomes (1-4). In mammalian cells, long-chain substrates are supposed to be degraded in peroxisomes and mediumor short-chain substrates in mitochondria (5). When microorganisms are cultivated on long-chain fatty acids or n-alkanes to be oxidized to fatty acids, the activity level of the /9-oxidation system increases; and the /J-oxidation system is detected only in peroxisomes (4, 6-9). However, the (3- oxidation system in eukaryotic microorganisms grown on short-chain fatty acids has not been studied in detail. Candida tropicalis is an invaluable microorganism in the study of fatty acid metabolism and biogenesis of peroxisomes. This yeast can utilize various carbon sources, such as glucose, acetate, propionate, and n-alkanes (10, 11). When it is cultivated on n-alkanes (CiO-Cu), the enzymes of the fatty acid /9-oxidation system are inducibly synthesized and specifically localized in peroxisomes (4, 6), which proliferate remarkably under these conditions (10). Fatty acids derived from n-alkanes are degraded via the peroxisomal /9-oxidation system. In the cells grown on glucose or acetate, induction of the enzymes of the /9-oxidation system and proliferation of peroxisomes were not observed (6, JO). In the propionate-grown cells, the activity levels of the enzymes of the /9-oxidation system increase above those in the glucose-grown cells and the volume of peroxisomes increases, though the physiological role of these enzymes in the propionate-grown cells is obscure (21, 12). In the present study, we have tried to cultivate this yeast on butyrate, which is the fatty acid of shortest chain-length to be degraded via the /9-oxidation system. We also examined 1
To whom correspondence should be addressed.
Vol. I l l , No. 6, 1992
the degree of peroxisome development, the activity level of the /9-oxidation enzymes, and their subcellular localization. Based on the results obtained, we discuss the difference in peroxisomal function in fatty acid /9-oxidation for shortchain-length substrates between yeast cells and mammalian cells. MATERIALS AND METHODS Cultivation of Yeast— C. tropicalis pK233 (ATCC 20336) was cultivated aerobically at 30'C in a medium containing glucose (16.5 g/liter), sodium acetate-3H2O (13.6g/liter), sodium propionate (10.0 g/liter), sodium butyrate (11.0 g/liter), or n-alkane mixture (CiO-Cu) (10 ml/liter) as the sole source of carbon and energy. The basic composition of the medium was described previously (11). In this study, 0.5 ml/liter of Tween 80 was added to each medium and the initial pH was adjusted to 6.0. The cultivation was carried out for 8 h on glucose and acetate, 22 h on propionate, 36 h on butyrate, and 17 h on alkanes. Preparation of Cell-Free Extracts and Subcellular Fractionation—Cell-free extracts were prepared by disintegrating the cells by sonication in 50 mM potassium phosphate buffer (pH7.2) containing 10% glycerol (11). Subcellular fractionation of the butyrate-grown cells was carried out in a similar manner to that of the propionategrown cells (12) except for the sucrose concentration used for density gradient centrifugation. Enzyme and Protein Assay—The activities of the enzymes of the fatty acid /9-oxidation system (acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and thiolase) were measured as described previously (6, 13). Thiolase was assayed using either acetoacetyl-CoA (for acetoacetyl-CoA thiolase activity) or 3-ketooctanoyl-CoA (for 3-ketoacyl-CoA thiolase activity) (14, 15). Catalase, cytochrome oxidase, and proteins were assayed by the methods described (16).
783
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M N
CM
B «vCW
J. Biochem.
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Peroxisomal fi-OxidaUon System in Butyrate-Grown C. tropicalis Electronmicroscopy—For electronmicroscopy, the butyrate-grown and glucose-grown cells were prefixed with 1% glutaraldehyde, and post-fixed with osmium tetraoxide as described previously {17). Immunoelectronmicroscopy of the butyrate-grown and alkane-grown cells was carried out as described (18, 19) using anti-(3-ketoacyl-CoA thiolase) antiserum. This antiserum was prepared using the enzyme purified from particulate fractions (20,000 Xg) containing peroxisomes and mitochondria (15). Chemicals—3-Ketooctanoyl-CoA was prepared enzymatically from octenoyl-C!oA (20), synthesized by the mixed anhydride method (21). Other chemicals were obtained from commercial sources. RESULTS Enzyme Activities in the Cells Grown on Various Carbon Sources— C. tropicalis was found to grow on butyrate as the sole source of carbon and energy, when the cultivation
Propionate Butyrate
conditions were optimized as described in "MATERIALS AND METHODS." The activities of catalase and the enzymes of the fatty acid /?-oxddation system in the cell-free extracts were compared among the cells grown on various carbon sources. As shown in Fig. 1, the activities of the enzymes of the fatty acid p- oxidation system were higher in the butyrate-grown and alkane-grown cells than in the glucosegrown, acetate-grown, and propionate-grown cells. The activities of enoyl-CoA hydratase, acetoacetyl -CoA thiolase, and 3-ketoacyl-CoA thiolase in the butyrate-grown cells were equal to or higher than those in the alkane-grown cells. The activity of acyl-CoA oxidase in the butyrategrown cells was lower than that in the alkane-grown cells, but it was higher than that in the cells grown on other carbon sources. The level of catalase activity in the butyrate-grown cells was comparable to that in the propionate-grown cells, but higher than that of the glucose- and acetate-grown cells. Electronmicroscopical Observation—The increase in the activity of catalase suggested the proliferation of peroxisomes in the butyrate-grown cells. This was confirmed by electronmicroscopy, as shown in Fig. 2, which revealed peroxisomes to be present profusely in the butyrate-grown cells but rarely in the glucose-grown cells. Subcellular Localization of Catalase and the Enzymes of the Fatty Acid ^-Oxidation System—As described above, catalase and the enzymes of the fatty acid /?- oxidation
Acjrl-CoA oxidase
Glucose
Butyrate
400 Enoyl-CoA hydntase
3-Ketoacyl-CoA
600
thiolase
(«!...„-'.
o Cr
30 1
15 | |
15
0 30
S--
20 •
Butyrate
3 0
100 200 300 Acetoacetyl-CoA thiolase (naol.nln"'.»g~')
Fig. 1. Specific activities of catalase and the enzymes of the fatty acid ^-oxidation system in C tropicalis cells grown on different carbon sources. (A) Catalase; (B) acyl-CoA oxidase; (C) enoyl-CoA hydratase; (D) 3-ketoacyl-CoA thiolase; (E) acetoacetylCoA thiolase. The profile of 3-hydroxyacyl-CoA dehydrogenase activity was the same as that of enoyl-CoA hydratase on the same multi-functional protein (28). Fig. 2. Electronmicrographs of the butyrate-grown (A) and glucose-grown cells (B) of C tropicalis. CM, cell membrane; CW, cell wall; M, mitochondrion; N, nucleus; P, peroxisome; V, vacuole. Bar, Vol. I l l , No. 6, 1992
20 • J0 ' 40 "41.3W2.5I 50 Sucrose concentration
(\)
20 ' 30 I *0 M l . 3 « 2 . S l Sucrose concentration
50 I (\)
Fig. 3. Particulate localization of emymes in the P, fraction (20,0OOXff pellet). The volume of each fraction was as follows: 1, 3.75 ml; 2-5, 2.5 ml each; 6, 1.25 ml. (A) Protein; (B) cytochrome oxidase; (C) catalase; (D) acyl-CoA oxidase; (E) enoyl-CoA hydratase; (F) 3-ketoacyl-CoA thiolase; (G) acetoacetyl-CoA thiolase.
786
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B
