J. Biochem. I l l , 20-24 (1992)
Mitochondrial Targeting Signal of Rat Liver Monoamine Oxidase B Is Located at Its Carboxy Terminus Jun-ya Mi torn a and Akio Ito Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, August 14, 1991
Monoamine oxidase B, a typical intrinsic protein of the outer mitochondrial membrane, has an uncleavable targeting signal and is inserted into the membrane without proteolytic maturation. To investigate the region responsible for targeting the enzyme to the outer mitochondrial membrane, various mutated proteins were expressed in cultured mammalian cells, and the distributions of the expressed proteins were analyzed by immunofluorescence microscopy and subcellular fractionation. Deletion of the carboxy-terminal 28 amino acids of monoamine oxidase B abolished the transfer of the enzyme to mitochondria, while the deletion of the amino-terminal 65 amino acids had no effect on the transfer to mitochondria. The existence of the targeting signal at the carboxy-terminal portion of the enzyme was confirmed by using hybrid proteins in which the amino- or carboxy-terminal portion of the enzyme was fused to the hydrophilic portion of cytochrome fe. The fused protein with the carboxy-terminal 29 amino acid residues of monoamine oxidase B was localized in mitochondria, whereas that with 10 amino acids remained in the cytoplasm. These results indicate that the targeting signal of monoamine oxidase B is present within its carboxy-terminal 29 amino acid residues.
Monoamine oxidase, which catalyzes the oxidative deamination of biogenic and xenobiotic amines, is a typical intrinsic protein of the outer mitochondrial membrane (1, 2). There are two molecular forms of this enzyme, A and B, which are coded by different genes and which have different substrate and inhibitor specificities (I). It has been reported that monoamine oxidase B was made on free polysomes, that the peptide synthesized in vitro in a cell-free translation system had the same apparent molecular size as the mature protein in the membrane, and that ubiquitin seemed to be involved in the in vitro insertion of the enzyme into the membrane (3-5). The amino acid sequences deduced from the cDNAs of rat (6, 7), bovine (8), and human (9, 10) monoamine oxidase A and B show that the enzymes have no typical structural feature common to the extension peptides of mitochondrial protein precursors, i.e., a long stretch of uncharged amino acid residues with intervention of positively charged residues, at their amino termini (11). In the present study, we intended to determine the region responsible for targeting and anchoring monoamine oxidase B to the outer mitochondrial membrane. Various mutated proteins with deletion in the amino- or carboxy-terminal portion and hybrid proteins, consisting of various lengths of the amino- or carboxy-terminal portion of the enzyme and the hydrophilic portion of cytochrome £%, were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The results showed that the signal for the targeting of monoamine oxidase B to the outer mitochondrial membrane is located at its carboxy-terminal region.
EXPERIMENTAL PROCEDURES
Materials—pSVL was obtained from Pharmacia LKB Biotechnology (Uppsala, Sweden). Restriction and modifying enzymes and pBluescript SK+ vectors were purchased from Takara Shuzo (Kyoto), Nippon Gene (Toyama), and Toyobo (Osaka). Immunostain HRP and FITC-conjugated goat anti-rabbit IgG were from Konica (Tokyo) and Cappel Products (U.S.A.), respectively. Cells and Media—COS-1, a simian kidney cell line transformed with simian virus 40 (SV40), was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum in an atmosphere of 10% C0 2 at 37*C. Escherichia coli strain DH5
Mitochondrial Targeting Signal of Rat Liver Monoamine Oxidase B Is Located at Its Carboxy Terminus Jun-ya Mi torn a and Akio Ito Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, August 14, 1991
Monoamine oxidase B, a typical intrinsic protein of the outer mitochondrial membrane, has an uncleavable targeting signal and is inserted into the membrane without proteolytic maturation. To investigate the region responsible for targeting the enzyme to the outer mitochondrial membrane, various mutated proteins were expressed in cultured mammalian cells, and the distributions of the expressed proteins were analyzed by immunofluorescence microscopy and subcellular fractionation. Deletion of the carboxy-terminal 28 amino acids of monoamine oxidase B abolished the transfer of the enzyme to mitochondria, while the deletion of the amino-terminal 65 amino acids had no effect on the transfer to mitochondria. The existence of the targeting signal at the carboxy-terminal portion of the enzyme was confirmed by using hybrid proteins in which the amino- or carboxy-terminal portion of the enzyme was fused to the hydrophilic portion of cytochrome fe. The fused protein with the carboxy-terminal 29 amino acid residues of monoamine oxidase B was localized in mitochondria, whereas that with 10 amino acids remained in the cytoplasm. These results indicate that the targeting signal of monoamine oxidase B is present within its carboxy-terminal 29 amino acid residues.
Monoamine oxidase, which catalyzes the oxidative deamination of biogenic and xenobiotic amines, is a typical intrinsic protein of the outer mitochondrial membrane (1, 2). There are two molecular forms of this enzyme, A and B, which are coded by different genes and which have different substrate and inhibitor specificities (I). It has been reported that monoamine oxidase B was made on free polysomes, that the peptide synthesized in vitro in a cell-free translation system had the same apparent molecular size as the mature protein in the membrane, and that ubiquitin seemed to be involved in the in vitro insertion of the enzyme into the membrane (3-5). The amino acid sequences deduced from the cDNAs of rat (6, 7), bovine (8), and human (9, 10) monoamine oxidase A and B show that the enzymes have no typical structural feature common to the extension peptides of mitochondrial protein precursors, i.e., a long stretch of uncharged amino acid residues with intervention of positively charged residues, at their amino termini (11). In the present study, we intended to determine the region responsible for targeting and anchoring monoamine oxidase B to the outer mitochondrial membrane. Various mutated proteins with deletion in the amino- or carboxy-terminal portion and hybrid proteins, consisting of various lengths of the amino- or carboxy-terminal portion of the enzyme and the hydrophilic portion of cytochrome £%, were expressed in cultured mammalian cells, and the subcellular localization of the expressed proteins was analyzed. The results showed that the signal for the targeting of monoamine oxidase B to the outer mitochondrial membrane is located at its carboxy-terminal region.
EXPERIMENTAL PROCEDURES
Materials—pSVL was obtained from Pharmacia LKB Biotechnology (Uppsala, Sweden). Restriction and modifying enzymes and pBluescript SK+ vectors were purchased from Takara Shuzo (Kyoto), Nippon Gene (Toyama), and Toyobo (Osaka). Immunostain HRP and FITC-conjugated goat anti-rabbit IgG were from Konica (Tokyo) and Cappel Products (U.S.A.), respectively. Cells and Media—COS-1, a simian kidney cell line transformed with simian virus 40 (SV40), was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum in an atmosphere of 10% C0 2 at 37*C. Escherichia coli strain DH5
