- Email: [email protected]
[7] Biosynthesis and intracellular sorting of mitochondrial forms of cytochrome P450
[7]
BIOSYNTHESIS OF MITOCHONDRIAL P 4 5 0 s
75
gested by protease even when microsomal membranes were present in the ceil-free mixtures. Interestingly, full-length proteins for the mutant forms of P450IIC2 and the hybrid were protected from protease, indicating that they were completely translocated across the membrane and did not contain any halt-transfer signals. In contrast, similar studies of rat P450IIB 1 in which a secretory protein signal peptide was substituted for the Nterminal sequence of the P450 showed that the protected protein was reduced in size. 2z The latter study is a nice demonstration of the use of protease digestion to show that a protein is partially translocated and is embedded in the membrane. Summary Analysis of P450 biosynthesis in cell-free systems has been useful in studying the mechanism of the targeting of P450 to the membrane (Table I). These techniques have allowed the demonstration that the N-terminal region of P450 is an uncleaved signal sequence for SRP-dependent membrane insertion. Lack of protection from protease indicated that most of the P450 is on the cytoplasmic side of the membrane. Substitution of basic amino acids at the P450 N terminus or replacing the N terminus of P450 with a secretory signal sequence results in partial or complete translocation across the membrane. This result indicates that at most one and probably none of the internal hydrophobic regions function as stop-transfer signals, which would be present if the protein spanned the membrane more than once. 22 S. Monier, P. Van Luc, G. Kreibich, D. D. Sabatini, and M. A d e s n i k , J. Cell Biol. 1M, 457 (1988).
[7] B i o s y n t h e s i s a n d I n t r a c e l l u l a r S o r t i n g o f M i t o c h o n d r i a l Forms of Cytochrome P450
By TSUNEO OMURA and AKIO ITO Introduction Mitochondrial forms of cytochrome P450 are encoded by nuclear genes, synthesized on cytoplasmic ribosomes as precursors, and imported into mitochondria posttranslationally. The precursor forms are larger than their mature counterparts found in mitochondria, the former having peptide extensions at the amino termini of the latter. The size of the extension METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.
76
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE P450SCC
: bovine
-39
+
adrenal
+
cortex
2
_
+
[7]
+++
_
~
+
MLARGLPLRSALVKACPPILSTVGEGWGHHRVGTGEGAGISTK
P450118
adrenal
: bovine
cortex
-24
+
+
....
3
+
+_
+
MALWAKARVRMAGPWLSLHEARLLGTRG
P450ST26
: rabbit
-36
+
¥
+ ....
liver 4 +
MAALGCARLRWALLG
+ P RVAGCGL
+
+
C P QGARAKAAI
PTAL PA ....
FIG. I. Extension peptides of the precursors of mitochondrial P450s. The amino-terminal
portions of the precursors of three mitochondrial P450s are shown. The extension peptides, whose sizes are indicated by the amino acid residue numbers at the amino termini, are underlined, and the processing points are marked with arrowheads. Charged amino acid residues are marked with + or - symbols.
peptides ranges from 24 amino acids for P450na (P450XIB1) to 39 amino acids for P450scc (P450XIA1). After import of the precursors into mitochondria, the extension peptides are cleaved from the mature portions by a processing protease in the matrix compartment, and the mature proteins are incorporated into the inner membrane. The mechanism of integration of the mature P450 molecules into the membrane is not yet elucidated. On the other hand, microsomal forms of cytochrome P450 are known to be incorporated into the membrane cotranslationaUy at the rough-surfaced portion of endoplasmic reticulum without any proteolytic processing of newly synthesized molecules. The extension peptides direct the import of the precursor proteins into mitochondria, and the information necessary for import seems to be contained in their amino-terminal 15-20 amino acid residues. Basic amino acid residues in this portion of the extension peptides seem to be essential for the import of the precursors. When this particular portion of an extension peptide is deleted or mutated, the modified precursor protein is no longer imported into the mitochondria. 1 Since the extension peptides are not present in the mature P450s purified from mitochondria, their primary structures have been deduced from the nucleotide sequences of the cloned P450s cDNAs. Figure 1 shows the amino acid sequences of the extension
I T. Kumamoto, K. Morohashi, A. Ito, and T. Omura, J. Biochem. (Tokyo) 102, 833 (1987).
[7]
BIOSYNTHESISOF MITOCHONDRIALP450s
77
peptides of three mitochondrial P450s, namely P45011t3, P450scc, and P450ST26 (P450XXVI). 2-4 The precursors of mitochondrial P450s synthesized in vitro are imported into isolated mitochondria. They are correctly processed, and the mature forms are incorporated into the inner membrane. 5 The import process is dependent on the membrane potential of the mitochondria and also on protein factors in the cytosol. Deenergization of mitochondria by uncouplers inhibits the import of the precursor proteins. Reticulocyte lysates contain the cytosolic factors necessary for the import reaction and are conveniently used in the translation of mitochondrial precursor proteins for in vitro import experiments. The in vitro import system seems to be reproducing the import reaction in the cells, but the latter is apparently much more efficient than the former. Detectable amounts of precursor proteins do not accumulate in the cells, whereas the import of some precursors in vitro is not complete, with some of the precursors remaining unimported after the incubation with mitochondria. Processing of the imported precursors to the mature forms is catalyzed by a processing protease(s) in mitochondria. The processing reaction is usually fast, and precursor proteins do not accumulate in the mitochondria. The processing protease(s) is apparently a metal protease(s) and is sensitive to heavy metal chelators. When mitochondria are incubated with ophenanthroline, a membrane-permeable metal chelator, before incubation with in vitro synthesized P450 precursors, the imported precursors accumulate in the matrix compartment. A soluble processing protease has been purified from rat liver mitochondria that processes the precursors of P450scc and P45011~ to the mature forms. 6 There is apparently no tissue selectivity in the import of precursor proteins into mitochondria prepared from various animal tissues. Liver mitochondria may be used in studying the in vitro import of steroidogenic P450s which are not expressed in the liver. Principles This chapter describes methods for studying the in vitro import of P450 precursors into mitochondria isolated from rat liver or bovine adrenal 2 K. Morohashi,Y. Fujii-Kuriyama,Y. Okada, K. Sogawa,T. Hirose, S. Inayama,and T. Omura, Proc. Natl. Acad. Sci. U.S.A. 81, 4647 (1984). 3 K. Morohashi,H. Yoshioka,O. Gotoh,Y. Okada, K. Yamamoto,T. Miyata,K. Sogawa, Y. Fujii-Kuriyama,and T. Omura, J. Biochem. (Tokyo) 102, 559 (1987). 4 S. Andersson,D. L. Davis, H. Dahlback, H. Jornvall, and D. W. Russel, J. Biol. Chem. 264, 8222 (1989). 5 W. J. Ou, A. Ito, K. Morohashi,Y. Fujii-Kuriyama,and T. Omura, J. Biochem. (Tokyo) 1110, 1287(1986). 6 W. J. Ou, A. Ito, H. Okazaki, and T. Omura, EMBO J. 8, 2605 (1989).
78
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[7]
cortex. The sorting of P450 precursors into mitochondria can also be studied with cultured mammalian cells transfected with P450 cDNAs,7 but the in vitro system is more convenient than the cultured cell system because the conditions for the import reaction may be easily manipulated. Yeast has also been frequently used in studying the import of various precursor proteins into mitochondria, but the import of P450 precursors has not been studied with yeast. Two cell-free translation systems, rabbit reticulocyte lysate and wheat germ extract, are commonly used in synthesizing various mammalian proteins in vitro. The reticulocyte lysate system is better suited for the synthesis of the precursor proteins of mitochondrial P450s because the lysate contains protein factors necessary for efficient import of the protein precursors. [35S]Methionine is usually used to label the translation products. The synthesized P450 precursors remain soluble in the translation mixture and are imported into mitochondria when the translation products are incubated with the isolated cell organelles. The import reaction depends on the integrity of the mitochondria and requires the inner membrane electrical potential. When total RNA or poly(A) ÷ RNA extracted from animal tissues is used for cell-free translation, the precursor and mature forms of the P450s must be separated from other labeled translation products by immunoprecipitation with specific antibodies before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. When a full-size cDNA of a mitochondrial P450 is available, it can be transcribed in vitro to give pure P450 mRNA, which is then translated with reticulocyte lysate to produce the P450 precursor protein. In the latter case, immunoprecipitation with P450 antibodies is not needed. By the use of the cDNA, modified P450 precursors may be easily prepared via manipulation of the cDNA to examine the structural requirements for correct import and processing of the precursor. ~ The in vitro import of P450 precursors into mitochondria is usually carried out at 250-30 °, and the efficiency of the import is determined by measuring the amounts of the mature forms by SDS-PAGE and fluorography after incubation of the import mixtures. The import reaction is slower at lower temperatures, and it does not proceed at 4°. The cell-free import experiments with isolated mitochondria and in vitro synthesized P450 precursors seem to be reproducing the intracellular sorting of the precursors to mitochondria. However, it is difficult to confirm the correct conformations of the P450 molecules incorporated into 7 K. Morohashi, Y. Nonaka, S. Kirita, O. Hatano, A. Takakusu, M. Okamoto, and T. Omura, J. Biochem. (Tokyo) 107, 635 (1990).
[7]
BIOSYNTHESISOF MITOCHONDR1ALP450s
79
the mitochondrial membrane in the cell-free system because the amounts of the in vitro synthesized P450 precursors are too small to measure the enzymatic activities after their conversion to the mature forms. Whether heme is correctly incorporated into the mature forms of P450s in the in vitro import experiments is also unclear.
Procedures
Import of Cytochrome P450 Synthesized from Total mRNA Preparation of Total RNA from Animal Tissues. Total RNA can be prepared from various animal tissues by extraction with strong denaturants such as SDS-phenol, guanidine thiocyanate, and guanidine hydrochloride. 8 About 1 mg of RNA is obtained from 1 g of bovine adrenal cortex. Poly(A) ÷ RNA is prepared from the total RNA by affinity chromatography on oligo(dT)-cellulose. 9 In Vitro Synthesis of Cytochrome P450 Precursors with Reticulocyte Lysate System.1° Total RNA preparations containing mRNAs of P450s are translated in a cell-free translation system to produce P450 precursor proteins. Rabbit reticulocyte lysate is usually used for the synthesis of the precursors of mitochondrial proteins because in vitro import of the precursors synthesized with wheat germ extracts into mitochondria is inefficient. Translation kits with nuclease-treated rabbit reticulocyte lysate are available from commerical manufacturers, but highly efficient lysates can be prepared from anemic rabbit blood. 11 The lysate should be stored at - 80° or in liquid nitrogen. Repeated freezing and thawing of the lysate should be avoided. The translation mixture (50/.d) contains 5 izl of 1 M potassium acetate, 5 ~1 of 10 mM magnesium acetate, 10/xl of translation cocktail, 15 ~1 of nuclease-treated rabbit reticulocyte lysate, 20/~g of total RNA [or 0.5/zg of poly(A) ÷ RNA], and 20-100 /~Ci of [35S]methionine (800-1200 Ci/ mmol). The translation cocktail consists of 150 mM H E P E S - K O H buffer (pH 7.5), 750/zg/ml creatine kinase, 200/.Lg/ml rabbit liver tRNA, 5 mM ATP, 2 mM GTP, 40 mM creatine phosphate, 150 mM each of 19 amino acids except methionine, 10 mM dithiothreitol (DTT), 3 mM spermidine, 8 R. J. MacDonald, G. H. Swift, A. E. Przybyla, and J. M. Chirgwin, this series, Vol. 152, p. 219. 9 H. Aviv and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 69, 1408 (1972). 10 A. Ito, T. Ogishima, W. Ou, T. Omura, H. Aoyagi, S. Lee, H. Mihara, and N. Izumiya, J. Biochem. (Tokyo) 98, 1571 (1985). 11 H. R. B. Pelham and R. J. Jackson. Eur. J. Biochem. 67, 247 (1976).
80
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[7]
and 200 units/ml rabbit liver or human placenta ribonuclease inhibitor. The translation reaction is carried out at 30° for 60-90 min. Preparation of Mitochondria from Animal Tissues for in Vitro Import Experiments. 1° Mitochondria for in vitro import experiments should have a high respiratory control ratio (4.0-6.0) and be structurally intact. Fresh rat livers are mildly homogenized in 9 volumes of fractionation medium consisting of 0.3 M sucrose, 10 mM H E P E S - K O H buffer (pH 7.5), 2 mM EDTA, and 2/~g/ml each of aprotinin, leupeptin, and pepstatin. The homogenate is centrifuged at 600 g for 10 min, and the supernatant is centrifuged further at 5000 g for 10 min. The tight pellet of mitochondria is suspended in 5 volumes of fractionation medium and centrifuged again using the same conditions. The washed mitochondria are suspended in import medium consisting of 0.3 M sucrose, 20 mM H E P E S - K O H (pH 7.5), and 2/~g/ml each of aprotinin, leupeptin, and pepstatin to a protein concentration of 5-20 mg/ml. Suspension of mitochondria is done using a loosely fitted Potter-type or Dounce-type homogenizer operated by hand. Mitochondria can be prepared by the same procedure from bovine adrenal cortex. Another isolation medium consisting of 0.22 M mannitol, 70 mM sucrose, and 5 mM Tris-HCl buffer (pH 7.5) may also be used in preparation of mitochondria from the tissues. In Vitro Import of P450 Precursors into Mitochondria.l°'12'13 The import reaction mixture contains 50/zl of the cell-free translation product and 250-500/~g protein of mitochondria in 500/~1 of import medium supplemented with 2 mM sodium succinate. The mixture is incubated at 30° for 30-60 min, then quickly chilled on ice. The mitochondria are then separated by centrifugation of the import mixture at 10,000 g for 5 min and solubilized with 1% SDS containing 10 mM EDTA. SDS and EDTA are also added to the supernatant. The precursor and mature forms of P450 are collected by immunoprecipitation from the solubilized mitochondria and the supernatant. The solubilized mixtures are heated in boiling water for 1 min and diluted with 10-20 volumes of 0.9% NaC1 containing 0.5% Triton X-100, 10 mM TrisHC1 (pH 7.5), and 2/~g/ml each of aprotinin, leupeptin, and pepstatin. Anti-P450 antibodies are added to the mixtures, which are then incubated for 30 min at 30°. Protein A-Sepharose or Staphylococcus aureus cells are added to the mixtures, incubated for 60 min at 30°, and recovered by centrifugation at 2000 g for 5 min. The precipitates are solubilized with SDS and analyzed by SDS-PAGE and fluorography. To confirm internalization of the precursor into mitochondria, the mito12 H. Ono and A. Ito, J. Biochem. (Tokyo) 95, 345 (1984). is T. Ogishima, Y. Okada, and T. Omura, J. Biochem. (Tokyo) 98, 781 (1985).
[7]
BIOSYNTHESIS OF MITOCHONDRIAL P450s
81
chondria are treated with a mixture of trypsin and chymotrypsin or with proteinase K, to digest any protein not internalized, before solubilization with SDS. After the import incubation, the precipitated mitochondria are suspended in 500 Izl of 0.3 M sucrose containing 20 mM H E P E S - K O H (pH 7.5), 20 /zM dinitrophenol, 1 mM EDTA, and 1 mM DTT, then treated with a 1 : 1 mixture of trypsin and chymotrypsin (5-25 tzg each) or proteinase K (2-15/zg) at 4° for 60 min. The protease treatment is terminated by addition of a 4-fold excess of soybean trypsin inhibitor and 1 mM phenylmethylsulfonyl fluoride (PMSF) for trypsin-chymotrypsin mixtures or 1 mM PMSF for proteinase K. Import of P450 Precursors Synthesized with in Vitro Transcription-Translation System 5 When full-size cDNA clones of P450s are available, P450 mRNAs can be synthesized in vitro using a DNA-dependent RNA polymerase. A P450 cDNA is inserted into the polylinker region of a pSP64 (or pSP65) vector, and then specific mRNA is transcribed from the SP6 plasmid linearized by cutting with an appropriate restriction enzyme downstream of the inserted cDNA. The transcription mixture (50/zl) contains 40 mM TrisHC1 (pH 7.5), 6 mM MgCI2, 2 mM spermidine, 10 mM DTT, 0.5 mM each of ATP, UTP, and CTP, 50/zM GTP, 0.5 mM mGpppG, 50 units of human placenta ribonuclease inhibitor, 0.01% bovine serum albumin, 5 tzg of linearized DNA, and 10 units of SP6 RNA polymerase. After incubation for 5 hr at 37°, product RNA is purified by phenol-chloroform extraction followed by ethanol precipitation from a 2 M ammonium acetate solution. Translation of the mRNA is carried out with the reticulocyte lysate system as described above. The RNA transcribed from 50-100 ng of the template DNA is used for 50/.d of the translation mixture. The import reaction may also be carried out as described above on a smaller scale (50-100/~1). The precursor and mature forms of the P450 are analyzed and quantitated by SDS-PAGE and fluorography without immunoprecipitation.
BIOSYNTHESIS OF MITOCHONDRIAL P 4 5 0 s
75
gested by protease even when microsomal membranes were present in the ceil-free mixtures. Interestingly, full-length proteins for the mutant forms of P450IIC2 and the hybrid were protected from protease, indicating that they were completely translocated across the membrane and did not contain any halt-transfer signals. In contrast, similar studies of rat P450IIB 1 in which a secretory protein signal peptide was substituted for the Nterminal sequence of the P450 showed that the protected protein was reduced in size. 2z The latter study is a nice demonstration of the use of protease digestion to show that a protein is partially translocated and is embedded in the membrane. Summary Analysis of P450 biosynthesis in cell-free systems has been useful in studying the mechanism of the targeting of P450 to the membrane (Table I). These techniques have allowed the demonstration that the N-terminal region of P450 is an uncleaved signal sequence for SRP-dependent membrane insertion. Lack of protection from protease indicated that most of the P450 is on the cytoplasmic side of the membrane. Substitution of basic amino acids at the P450 N terminus or replacing the N terminus of P450 with a secretory signal sequence results in partial or complete translocation across the membrane. This result indicates that at most one and probably none of the internal hydrophobic regions function as stop-transfer signals, which would be present if the protein spanned the membrane more than once. 22 S. Monier, P. Van Luc, G. Kreibich, D. D. Sabatini, and M. A d e s n i k , J. Cell Biol. 1M, 457 (1988).
[7] B i o s y n t h e s i s a n d I n t r a c e l l u l a r S o r t i n g o f M i t o c h o n d r i a l Forms of Cytochrome P450
By TSUNEO OMURA and AKIO ITO Introduction Mitochondrial forms of cytochrome P450 are encoded by nuclear genes, synthesized on cytoplasmic ribosomes as precursors, and imported into mitochondria posttranslationally. The precursor forms are larger than their mature counterparts found in mitochondria, the former having peptide extensions at the amino termini of the latter. The size of the extension METHODS IN ENZYMOLOGY, VOL. 206
Copyright © 1991by AcademicPress, Inc. All rights of reproduction in any form reserved.
76
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE P450SCC
: bovine
-39
+
adrenal
+
cortex
2
_
+
[7]
+++
_
~
+
MLARGLPLRSALVKACPPILSTVGEGWGHHRVGTGEGAGISTK
P450118
adrenal
: bovine
cortex
-24
+
+
....
3
+
+_
+
MALWAKARVRMAGPWLSLHEARLLGTRG
P450ST26
: rabbit
-36
+
¥
+ ....
liver 4 +
MAALGCARLRWALLG
+ P RVAGCGL
+
+
C P QGARAKAAI
PTAL PA ....
FIG. I. Extension peptides of the precursors of mitochondrial P450s. The amino-terminal
portions of the precursors of three mitochondrial P450s are shown. The extension peptides, whose sizes are indicated by the amino acid residue numbers at the amino termini, are underlined, and the processing points are marked with arrowheads. Charged amino acid residues are marked with + or - symbols.
peptides ranges from 24 amino acids for P450na (P450XIB1) to 39 amino acids for P450scc (P450XIA1). After import of the precursors into mitochondria, the extension peptides are cleaved from the mature portions by a processing protease in the matrix compartment, and the mature proteins are incorporated into the inner membrane. The mechanism of integration of the mature P450 molecules into the membrane is not yet elucidated. On the other hand, microsomal forms of cytochrome P450 are known to be incorporated into the membrane cotranslationaUy at the rough-surfaced portion of endoplasmic reticulum without any proteolytic processing of newly synthesized molecules. The extension peptides direct the import of the precursor proteins into mitochondria, and the information necessary for import seems to be contained in their amino-terminal 15-20 amino acid residues. Basic amino acid residues in this portion of the extension peptides seem to be essential for the import of the precursors. When this particular portion of an extension peptide is deleted or mutated, the modified precursor protein is no longer imported into the mitochondria. 1 Since the extension peptides are not present in the mature P450s purified from mitochondria, their primary structures have been deduced from the nucleotide sequences of the cloned P450s cDNAs. Figure 1 shows the amino acid sequences of the extension
I T. Kumamoto, K. Morohashi, A. Ito, and T. Omura, J. Biochem. (Tokyo) 102, 833 (1987).
[7]
BIOSYNTHESISOF MITOCHONDRIALP450s
77
peptides of three mitochondrial P450s, namely P45011t3, P450scc, and P450ST26 (P450XXVI). 2-4 The precursors of mitochondrial P450s synthesized in vitro are imported into isolated mitochondria. They are correctly processed, and the mature forms are incorporated into the inner membrane. 5 The import process is dependent on the membrane potential of the mitochondria and also on protein factors in the cytosol. Deenergization of mitochondria by uncouplers inhibits the import of the precursor proteins. Reticulocyte lysates contain the cytosolic factors necessary for the import reaction and are conveniently used in the translation of mitochondrial precursor proteins for in vitro import experiments. The in vitro import system seems to be reproducing the import reaction in the cells, but the latter is apparently much more efficient than the former. Detectable amounts of precursor proteins do not accumulate in the cells, whereas the import of some precursors in vitro is not complete, with some of the precursors remaining unimported after the incubation with mitochondria. Processing of the imported precursors to the mature forms is catalyzed by a processing protease(s) in mitochondria. The processing reaction is usually fast, and precursor proteins do not accumulate in the mitochondria. The processing protease(s) is apparently a metal protease(s) and is sensitive to heavy metal chelators. When mitochondria are incubated with ophenanthroline, a membrane-permeable metal chelator, before incubation with in vitro synthesized P450 precursors, the imported precursors accumulate in the matrix compartment. A soluble processing protease has been purified from rat liver mitochondria that processes the precursors of P450scc and P45011~ to the mature forms. 6 There is apparently no tissue selectivity in the import of precursor proteins into mitochondria prepared from various animal tissues. Liver mitochondria may be used in studying the in vitro import of steroidogenic P450s which are not expressed in the liver. Principles This chapter describes methods for studying the in vitro import of P450 precursors into mitochondria isolated from rat liver or bovine adrenal 2 K. Morohashi,Y. Fujii-Kuriyama,Y. Okada, K. Sogawa,T. Hirose, S. Inayama,and T. Omura, Proc. Natl. Acad. Sci. U.S.A. 81, 4647 (1984). 3 K. Morohashi,H. Yoshioka,O. Gotoh,Y. Okada, K. Yamamoto,T. Miyata,K. Sogawa, Y. Fujii-Kuriyama,and T. Omura, J. Biochem. (Tokyo) 102, 559 (1987). 4 S. Andersson,D. L. Davis, H. Dahlback, H. Jornvall, and D. W. Russel, J. Biol. Chem. 264, 8222 (1989). 5 W. J. Ou, A. Ito, K. Morohashi,Y. Fujii-Kuriyama,and T. Omura, J. Biochem. (Tokyo) 1110, 1287(1986). 6 W. J. Ou, A. Ito, H. Okazaki, and T. Omura, EMBO J. 8, 2605 (1989).
78
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[7]
cortex. The sorting of P450 precursors into mitochondria can also be studied with cultured mammalian cells transfected with P450 cDNAs,7 but the in vitro system is more convenient than the cultured cell system because the conditions for the import reaction may be easily manipulated. Yeast has also been frequently used in studying the import of various precursor proteins into mitochondria, but the import of P450 precursors has not been studied with yeast. Two cell-free translation systems, rabbit reticulocyte lysate and wheat germ extract, are commonly used in synthesizing various mammalian proteins in vitro. The reticulocyte lysate system is better suited for the synthesis of the precursor proteins of mitochondrial P450s because the lysate contains protein factors necessary for efficient import of the protein precursors. [35S]Methionine is usually used to label the translation products. The synthesized P450 precursors remain soluble in the translation mixture and are imported into mitochondria when the translation products are incubated with the isolated cell organelles. The import reaction depends on the integrity of the mitochondria and requires the inner membrane electrical potential. When total RNA or poly(A) ÷ RNA extracted from animal tissues is used for cell-free translation, the precursor and mature forms of the P450s must be separated from other labeled translation products by immunoprecipitation with specific antibodies before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. When a full-size cDNA of a mitochondrial P450 is available, it can be transcribed in vitro to give pure P450 mRNA, which is then translated with reticulocyte lysate to produce the P450 precursor protein. In the latter case, immunoprecipitation with P450 antibodies is not needed. By the use of the cDNA, modified P450 precursors may be easily prepared via manipulation of the cDNA to examine the structural requirements for correct import and processing of the precursor. ~ The in vitro import of P450 precursors into mitochondria is usually carried out at 250-30 °, and the efficiency of the import is determined by measuring the amounts of the mature forms by SDS-PAGE and fluorography after incubation of the import mixtures. The import reaction is slower at lower temperatures, and it does not proceed at 4°. The cell-free import experiments with isolated mitochondria and in vitro synthesized P450 precursors seem to be reproducing the intracellular sorting of the precursors to mitochondria. However, it is difficult to confirm the correct conformations of the P450 molecules incorporated into 7 K. Morohashi, Y. Nonaka, S. Kirita, O. Hatano, A. Takakusu, M. Okamoto, and T. Omura, J. Biochem. (Tokyo) 107, 635 (1990).
[7]
BIOSYNTHESISOF MITOCHONDR1ALP450s
79
the mitochondrial membrane in the cell-free system because the amounts of the in vitro synthesized P450 precursors are too small to measure the enzymatic activities after their conversion to the mature forms. Whether heme is correctly incorporated into the mature forms of P450s in the in vitro import experiments is also unclear.
Procedures
Import of Cytochrome P450 Synthesized from Total mRNA Preparation of Total RNA from Animal Tissues. Total RNA can be prepared from various animal tissues by extraction with strong denaturants such as SDS-phenol, guanidine thiocyanate, and guanidine hydrochloride. 8 About 1 mg of RNA is obtained from 1 g of bovine adrenal cortex. Poly(A) ÷ RNA is prepared from the total RNA by affinity chromatography on oligo(dT)-cellulose. 9 In Vitro Synthesis of Cytochrome P450 Precursors with Reticulocyte Lysate System.1° Total RNA preparations containing mRNAs of P450s are translated in a cell-free translation system to produce P450 precursor proteins. Rabbit reticulocyte lysate is usually used for the synthesis of the precursors of mitochondrial proteins because in vitro import of the precursors synthesized with wheat germ extracts into mitochondria is inefficient. Translation kits with nuclease-treated rabbit reticulocyte lysate are available from commerical manufacturers, but highly efficient lysates can be prepared from anemic rabbit blood. 11 The lysate should be stored at - 80° or in liquid nitrogen. Repeated freezing and thawing of the lysate should be avoided. The translation mixture (50/.d) contains 5 izl of 1 M potassium acetate, 5 ~1 of 10 mM magnesium acetate, 10/xl of translation cocktail, 15 ~1 of nuclease-treated rabbit reticulocyte lysate, 20/~g of total RNA [or 0.5/zg of poly(A) ÷ RNA], and 20-100 /~Ci of [35S]methionine (800-1200 Ci/ mmol). The translation cocktail consists of 150 mM H E P E S - K O H buffer (pH 7.5), 750/zg/ml creatine kinase, 200/.Lg/ml rabbit liver tRNA, 5 mM ATP, 2 mM GTP, 40 mM creatine phosphate, 150 mM each of 19 amino acids except methionine, 10 mM dithiothreitol (DTT), 3 mM spermidine, 8 R. J. MacDonald, G. H. Swift, A. E. Przybyla, and J. M. Chirgwin, this series, Vol. 152, p. 219. 9 H. Aviv and P. Leder, Proc. Natl. Acad. Sci. U.S.A. 69, 1408 (1972). 10 A. Ito, T. Ogishima, W. Ou, T. Omura, H. Aoyagi, S. Lee, H. Mihara, and N. Izumiya, J. Biochem. (Tokyo) 98, 1571 (1985). 11 H. R. B. Pelham and R. J. Jackson. Eur. J. Biochem. 67, 247 (1976).
80
MUTAGENESIS, MODIFICATION, PROTEIN STRUCTURE
[7]
and 200 units/ml rabbit liver or human placenta ribonuclease inhibitor. The translation reaction is carried out at 30° for 60-90 min. Preparation of Mitochondria from Animal Tissues for in Vitro Import Experiments. 1° Mitochondria for in vitro import experiments should have a high respiratory control ratio (4.0-6.0) and be structurally intact. Fresh rat livers are mildly homogenized in 9 volumes of fractionation medium consisting of 0.3 M sucrose, 10 mM H E P E S - K O H buffer (pH 7.5), 2 mM EDTA, and 2/~g/ml each of aprotinin, leupeptin, and pepstatin. The homogenate is centrifuged at 600 g for 10 min, and the supernatant is centrifuged further at 5000 g for 10 min. The tight pellet of mitochondria is suspended in 5 volumes of fractionation medium and centrifuged again using the same conditions. The washed mitochondria are suspended in import medium consisting of 0.3 M sucrose, 20 mM H E P E S - K O H (pH 7.5), and 2/~g/ml each of aprotinin, leupeptin, and pepstatin to a protein concentration of 5-20 mg/ml. Suspension of mitochondria is done using a loosely fitted Potter-type or Dounce-type homogenizer operated by hand. Mitochondria can be prepared by the same procedure from bovine adrenal cortex. Another isolation medium consisting of 0.22 M mannitol, 70 mM sucrose, and 5 mM Tris-HCl buffer (pH 7.5) may also be used in preparation of mitochondria from the tissues. In Vitro Import of P450 Precursors into Mitochondria.l°'12'13 The import reaction mixture contains 50/zl of the cell-free translation product and 250-500/~g protein of mitochondria in 500/~1 of import medium supplemented with 2 mM sodium succinate. The mixture is incubated at 30° for 30-60 min, then quickly chilled on ice. The mitochondria are then separated by centrifugation of the import mixture at 10,000 g for 5 min and solubilized with 1% SDS containing 10 mM EDTA. SDS and EDTA are also added to the supernatant. The precursor and mature forms of P450 are collected by immunoprecipitation from the solubilized mitochondria and the supernatant. The solubilized mixtures are heated in boiling water for 1 min and diluted with 10-20 volumes of 0.9% NaC1 containing 0.5% Triton X-100, 10 mM TrisHC1 (pH 7.5), and 2/~g/ml each of aprotinin, leupeptin, and pepstatin. Anti-P450 antibodies are added to the mixtures, which are then incubated for 30 min at 30°. Protein A-Sepharose or Staphylococcus aureus cells are added to the mixtures, incubated for 60 min at 30°, and recovered by centrifugation at 2000 g for 5 min. The precipitates are solubilized with SDS and analyzed by SDS-PAGE and fluorography. To confirm internalization of the precursor into mitochondria, the mito12 H. Ono and A. Ito, J. Biochem. (Tokyo) 95, 345 (1984). is T. Ogishima, Y. Okada, and T. Omura, J. Biochem. (Tokyo) 98, 781 (1985).
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BIOSYNTHESIS OF MITOCHONDRIAL P450s
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chondria are treated with a mixture of trypsin and chymotrypsin or with proteinase K, to digest any protein not internalized, before solubilization with SDS. After the import incubation, the precipitated mitochondria are suspended in 500 Izl of 0.3 M sucrose containing 20 mM H E P E S - K O H (pH 7.5), 20 /zM dinitrophenol, 1 mM EDTA, and 1 mM DTT, then treated with a 1 : 1 mixture of trypsin and chymotrypsin (5-25 tzg each) or proteinase K (2-15/zg) at 4° for 60 min. The protease treatment is terminated by addition of a 4-fold excess of soybean trypsin inhibitor and 1 mM phenylmethylsulfonyl fluoride (PMSF) for trypsin-chymotrypsin mixtures or 1 mM PMSF for proteinase K. Import of P450 Precursors Synthesized with in Vitro Transcription-Translation System 5 When full-size cDNA clones of P450s are available, P450 mRNAs can be synthesized in vitro using a DNA-dependent RNA polymerase. A P450 cDNA is inserted into the polylinker region of a pSP64 (or pSP65) vector, and then specific mRNA is transcribed from the SP6 plasmid linearized by cutting with an appropriate restriction enzyme downstream of the inserted cDNA. The transcription mixture (50/zl) contains 40 mM TrisHC1 (pH 7.5), 6 mM MgCI2, 2 mM spermidine, 10 mM DTT, 0.5 mM each of ATP, UTP, and CTP, 50/zM GTP, 0.5 mM mGpppG, 50 units of human placenta ribonuclease inhibitor, 0.01% bovine serum albumin, 5 tzg of linearized DNA, and 10 units of SP6 RNA polymerase. After incubation for 5 hr at 37°, product RNA is purified by phenol-chloroform extraction followed by ethanol precipitation from a 2 M ammonium acetate solution. Translation of the mRNA is carried out with the reticulocyte lysate system as described above. The RNA transcribed from 50-100 ng of the template DNA is used for 50/.d of the translation mixture. The import reaction may also be carried out as described above on a smaller scale (50-100/~1). The precursor and mature forms of the P450 are analyzed and quantitated by SDS-PAGE and fluorography without immunoprecipitation.