The use of gene fusions to determine the topology of all of the subunits of the cytochrome o terminal oxidase complex of Escherichia coli

The cytochrome o terminal oxidase complex is a component of the aerobic respiratory chain of Escherichia coli. This enzyme catalyzes the oxidation of ubiquinol-8 to ubiquinone-8 within the cytoplasmic membrane and the concomitant reduction of O2 to H2O. The hydropathy profiles of the deduced amino acid sequences suggest that all five of the gene products of the cyo operon contain multiple membrane-spanning helical segments. The goal of this work was to obtain experimental evidence for the topology of the five gene products in the cytoplasmic membrane by using the technique of gene fusions. A number of random gene fusions were generated in vitro encoding hybrid proteins in which the amino-terminal portion was provided by the subunit of interest and the carboxyl-terminal portion by one of two sensor proteins, alkaline phosphatase lacking its signal sequence or beta-galactosidase. Results obtained are self-consistent, and topological models are proposed for all of the five gene products encoded by the cyo operon. Based on the sequence similarities with subunits of the aa3-type cytochrome c oxidases, the experimental evidence obtained here can be used to infer topological models for the mitochondrial encoded subunits of the eukaryotic cytochrome c oxidases.     

Expression of cyoA and cyoB demonstrates that the CO-binding heme component of the Escherichia coli cytochrome o complex is in subunit I

The cytochrome o complex of the Escherichia coli aerobic respiratory chain is a ubiquinol oxidase. The enzyme consists of at least four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and contains two heme b prosthetic groups (b555 and b562) plus copper. The sequence of the cyo operon, encoding the subunits of the oxidase, reveals five open reading frames, cyoABCDE. This paper describes results obtained by expressing independently cyoA and cyoB in the absence of the other subunits of the complex. Polyclonal antibodies which react with subunits I and II of the purified oxidase demonstrate that cyoA and cyoB correspond to subunit II and subunit I, respectively, of the complex. These subunits are stably inserted into the membrane when expressed. Furthermore, expression of cyoB (subunit I) results in elevated heme levels in the membrane. Reduced-minus-oxidized spectra suggest that the cytochrome b555 component is present but that the cytochrome b562 component is not. This heme component is shown to bind to CO, as it does in the intact enzyme. Hence, subunit I alone is sufficient for the assembly of the stable CO-binding heme component of this oxidase.     

Characterization of the cytochrome d terminal oxidase complex of Escherichia coli using polyclonal and monoclonal antibodies

The cytochrome d terminal oxidase complex is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. Previous work has shown by dodecyl sulfate-polyacrylamide gel electrophoresis that this enzyme contains two subunits (I and II) and three cytochrome components, b558 , a1, and d. Reconstitution studies have demonstrated that the enzyme functions as a ubiquinol-8 oxidase and catalyzes an electrogenic reaction, i.e. turnover is accompanied by a charge separation across the membrane bilayer. In this paper, monoclonal and polyclonal antibodies were used to obtain structural information about the cytochrome d complex. It is shown that antibodies directed against subunit I effectively inhibit ubiquinol-1 oxidation by the purified enzyme in detergent, whereas antibodies which bind to subunit II have no effect on quinol oxidation. The oxidation rate of N,N,N',N'-tetramethyl-p-phenylenediamine, in contrast, is unaffected by antisubunit I antibodies, but is inhibited by antibodies against subunit II. It is concluded that the quinol oxidation site is on subunit I, previously shown to be the cytochrome b558 component of the complex, and that N,N,N',N'-tetramethyl-p-phenylenediamine oxidation occurs at a secondary site on subunit II. The antibodies were also used to analyze the results of a protein cross-linking experiment. Dimethyl suberimidate was used to cross-link the subunits of purified, solubilized oxidase. Immunoblot analysis of the products of this cross-linking clearly indicate that subunit II probably exists as a dimer within the complex. Finally, it is shown that the purified enzyme contains tightly bound lipopolysaccharide. This was revealed after discovering that one of the monoclonal antibodies raised against the purified complex is actually directed against lipopolysaccharide. The significance of this finding is not known.     

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.     

Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.     

Subunit II of cytochrome c oxidase in Chlamydomonad algae is a heterodimer encoded by two independent nuclear genes

The mitochondrial genomes of Chlamydomonad algae lack the cox2 gene that encodes the essential subunit COX II of cytochrome c oxidase. COX II is normally a single polypeptide encoded by a single mitochondrial gene. In this work we cloned two nuclear genes encoding COX II from both Chlamydomonas reinhardtii and Polytomella sp. The cox2a gene encodes a protein, COX IIA, corresponding to the N-terminal portion of subunit II of cytochrome c oxidase, and the cox2b gene encodes COX IIB, corresponding to the C-terminal region. The cox2a and cox2b genes are located in the nucleus and are independently transcribed into mRNAs that are translated into separate polypeptides. These two proteins assemble with other cytochrome c oxidase subunits in the inner mitochondrial membrane to form the mature multi-subunit complex. We propose that during the evolution of the Chlorophyte algae, the cox2 gene was divided into two mitochondrial genes that were subsequently transferred to the nucleus. This event was evolutionarily distinct from the transfer of an intact cox2 gene to the nucleus in some members the Leguminosae plant family.     

Heme A is not essential for assembly of the subunits of cytochrome c oxidase of Rhodobacter sphaeroides

The aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides, a proteobacterium of the alpha subgroup, is structurally similar to the core subunits of the terminal oxidase in the mitochondrial electron transport chain. Subunit I, the product of the coxI gene, normally binds two heme A molecules. A deletion of cox10, the gene for the farnesyltransferase required for heme A synthesis, did not prevent high level accumulation of subunit I in the cytoplasmic membrane. Thus, subunit I can be expressed and stably inserted into the cytoplasmic membrane in the absence of heme A. Aposubunit I was purified via affinity chromatography to a polyhistidine tag. Copurification of subunits II and III with aposubunit I indicated that assembly of the core oxidase complex occurred without the binding of heme A. In addition to formation of the apooxidase containing all three large structural proteins, CoxI-II and CoxI-III heterodimers were isolated from cox10 deletion strains harboring expression plasmids with coxI and coxII or with coxI and coxIII, respectively. This demonstrated that subunit assembly of the apoenzyme was not an inherently ordered or sequential process. Thus, multiple paths must be considered for understanding the assembly of this integral membrane metalloprotein complex.     

Deletion of the COX7 gene in Saccharomyces cerevisiae reveals a role for cytochrome c oxidase subunit VII In assembly of remaining subunits

Cytochrome c oxidase from Saccharomyces cerevisiae is composed of nine subunits. Subunits I, II and III are products of mitochondrial genes, while subunits IV, V, VI, VII, VIIa and VIII are products of nuclear genes. To investigate the role of cytochrome c oxidase subunit VII in biogenesis or functioning of the active enzyme complex, a null mutation in the COX7 gene, which encodes subunit VII, was generated, and the resulting cox7 mutant strain was characterized. The strain lacked cytochrome c oxidase activity and haem a/a3 spectra. The strain also lacked subunit VII, which should not be synthesized owing to the nature of the cox7 mutation generated in this strain. The amounts of remaining cytochrome c oxidase subunits in the cox7 mutant were examined. Accumulation of subunit I, which is the product of the mitochondrial COX1 gene, was found to be decreased relative to other mitochondrial translation products. Results of pulse-chase analysis of mitochondrial translation products are consistent with either a decreased rate of translation of COX1 mRNA or a very rapid rate of degradation of nascent subunit I. The synthesis, stability or mitochondrial localization of the remaining nuclear-encoded cytochrome c oxidase subunits were not substantially affected by the absence of subunit VII. To investigate whether assembly of any of the remaining cytochrome c oxidase subunits is impaired in the mutant strain, the association of the mitochondrial-encoded subunits I, II and III with the nuclear-encoded subunit IV was investigated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly cross-linked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700: and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti-I immunoglobulin did not inhibit the activity. The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.     

Electrophoretic behaviour of yeast mitochondrial translation products.

We have studied the mobility of yeast mitochondrial translation products during electrophoresis on polyacrylamide gels of different composition and found that these polypeptides can be divided into two groups. One, to which subunit II of cytochrome c oxidase belongs, behaves normal as all water-soluble reference proteins. The other, to which cytochrome b and subunits I and III of cytochrome c oxidase belong, shows a free electrophoretic mobility about twice as fast as the first group. Conditions have been found to separate cytochrome c1 from cytochrome b.     

A critical evaluation of the hydropathy profile of membrane proteins

New membrane-preference scales are introduced for categories of membrane proteins with different functions. A statistical analysis is carried out with several scales to verify the relative accuracy in the prediction of the transmembrane segments of polytopic membrane proteins. The correlation between some of the scales most used and those calculated here provides criteria for selecting the most appropriate methods for a given type of protein. The parameters used in the evaluation of the hydropathy profiles have been carefully ascertained in order to develop a reliable methodology for hydropathy analysis. Finally, an integrated hydropathy analysis using different methods has been applied to several sequences of related proteins. The above analysis indicates that (a) microsomal cytochrome P450 contains only one hydrophobic region at the N-terminus that is consistently predicted to transverse the membrane: (b) only four of the six or seven putative transmembrane helices of cytochrome oxidase subunit III are predicted and correspond to helices I, III, V and VI of the previous nomenclature; (c) the product of the mitochondrial ATPase-6 gene (or the chloroplast ATPase-IV gene) of F0-F1-ATPase shows that helix IV is not consistently predicted to traverse the membrane, suggesting a four-helix model for this family of proteins.     

Studies on the assembly of cytochrome oxidase in isolated rat hepatocytes.

1. The assembly of rat liver cytochrome oxidase was studied in isolated hepatocytes and isolated liver mitochondria labelled with L-[35S]methionine. 2. Labelled subunits II and III appeared in the immunoabsorbed holoenzyme within minutes after the initiation of a pulse label. In contrast, labelled subunit I appeared in immunoabsorbed holoenzyme only after a subsequent 2 h chase or after an additional 2 h of labelling. Subunit I was heavily labelled, however, in intact mitochondria after 10 min. 3. A similar pattern of labelling was observed in holo-cytochrome oxidase which was chemically isolated by a small scale procedure adapted for this purpose. The appearance of subunit I in the holoenzyme was delayed for 1.5-2 h after a 60 min pulse with labelled methionine. 4. Incubation of hepatocytes for 4 h in the presence of cycloheximide had no effect on the labelling pattern described above. 5. Methods were developed in which newly translated, presumably unassembled, subunits of cytochrome oxidase could be separated from the holoenzyme by fractionation in Triton X-114. Short-term pulse experiments indicate that subunits II and III are associated with the holoenzyme fraction immediately after their completion, whereas subunit I is not. 6. The data are consistent with a model in which cytochrome oxidase assembly is viewed as an ordered and sequential event.     

Evidence for cytochrome oxidase subunit I and a cytochrome c--subunit II fused protein in the cytochrome 'c1aa3' of Thermus thermophilus. How old is cytochrome oxidase?

The terminal cytochrome c1aa3 of the respiratory chain of Thermus thermophilus has been isolated and purified to homogeneity by a novel procedure. The two subunit proteins (55 and 33 kDa) have been characterized chemically. Computer searches with partial amino acid sequences obtained from both subunits show that the larger subunit belongs to the cytochrome oxidase subunit I protein family while the smaller covalently heme-binding subunit is not a cytochrome c1 but appears to be a fused protein between cytochrome c and cytochrome oxidase subunit II. With respect to the 16-S rRNA-derived phylogeny of procaryotes, the results show that the genetic information for an O2-reacting cytochrome oxidase (EC 1.9.3.1) existed already in early eubacteria.     

Labeling of cytochrome c oxidase with [35S]diazobenzenesulfonate. Orientation of this electron transfer complex in the inner mitochondrial membrane.

Isolated cytochrome c oxidase was fractionated by native-gel electrophoresis in Triton X-100, and a preparation of enzyme almost completely free of the usual impurities was recovered. This fraction was used to generate antibodies specific to cytochrome c oxidase. These antibodies inhibited cytochrome c oxidase activity rapidly and completely and immunoprecipitated an enzyme containing seven different subunits from detergent-solubilized mitochondria or submitochondrial particles. Reaction of detergent-solubilized cytochrome c oxidase with [35S]diazobenzenesulfonate labeled all seven subunits although I and VI were much less reactive than the other five components. When cytochrome c oxidase was immunoprecipitated from mitochondria which had been reacted with [35S]DABS, subunits II and III were the only components labeled. When the complex was immunoprecipitated from labeled submitochondrial particles, II, III, IV, V, and VII were all labeled. Polypeptides I and VI were not labeled from either side of the membrane. These results confirm earlier studies which showed that cytochrome c oxidase spans the mitochondrial inner membrane and is asymmetrically arranged across this permeability barrier.     

Cytochrome c oxidase subunits in contact with phospholipids. Hydrophobic photolabeling with azidophospholipids.

The technique of photolabeling of membrane proteins with arylazidophospholipids was applied to cytochrome c oxidase. The "deep" and "shallow" labels employed reacted with all subunits of cytochrome c oxidase except V and VI: Subunits I, III, and VII were heavily labeled, Subunit II was labeled to a lesser extent, and Subunit IV was poorly labeled. Subunit I was labeled more by the deep label and Subunit VII by the shallow one. The other subunits were equally labeled by the two probes. This technique has revealed what subunits of cytochrome c oxidase interact with the lipid and their approximate position in the membrane.     

Asymmetrical Evolution of Cytochrome bd Subunits

Functionally linked genes generally evolve at similar rates and the knowledge of this particular feature of genomic evolution has been used as the basis for the phylogenetic profiling method. We illustrate here an exception to this rule in the evolution of the cytochrome bd complex. This is a two-component oxidase complex, with the subunits I and II known to be widely present in bacteria. The subunits within the cytochrome bd complex are under the same evolutionary pressure and most likely behave in the same evolutionary manner. However, the sequence similarity of genes encoding subunit II varies considerably across species. Genes encoding subunit II evolve 1.2 times faster on most of the branches of their phylogeny than subunit I genes. Furthermore, the genes encoding subunit II in Oceanobacillus iheyensis, Bacillus halodurans, and Staphylococcus species do not have detectable homologues within E. coli due to their large divergence. Together, the two subunits of cytochrome bd reveal an interesting example of an asymmetric pattern of evolutionary change. [Reviewing Editor: Dr. Brian Morton]     

Mechanism of increase in cytochrome c oxidase activity in sweet potato root tissue during aging of slices

The mechanism of an increase in cytochrome c oxidase [EC 1.9.3.1] activity during aging of sliced sweet potato root tissue was investigated with antibiotics and antibody to the purified enzyme. 1. The increase in cytochrome c oxidase activity was inhibited by chloramphenicol but not by cycloheximide. 2. Cytochrome c oxidase purified from wounded tissue was identical with that from intact tissue as judged by the subunit composition, sedimentation velocity, absorption spectrum, antigenicity, and activity per heme a. 3. An increase in the amount of cytochrome c oxidase protein took place during aging of slices. 4. Sweet potato cytochrome c oxidase consists of five subunits. When slices were aged in the presence of [3H]leucine, the three larger subunits (I, II, and III) of cytochrome c oxidase were labeled, while no radioactivity was incorporated into the other two subunits, IV and V. The results indicate that the increase in cytochrome c oxidase activity is due to an increase in the amount of the enzyme protein. We propose that excess amounts of subunits derived from the cytoplasm of the enzyme are present in intact tissue and are assembled with subunits of mitochondrial origin to form the holoenzyme after wounding of tissue.     

Nuclear functions required for cytochrome c oxidase biogenesis in Saccharomyces cerevisiae. Characterization of mutants in 34 complementation groups

To identify nuclear functions required for cytochrome c oxidase biogenesis in yeast, recessive nuclear mutants that are deficient in cytochrome c oxidase were characterized. In complementation studies, 55 independently isolated mutants were placed into 34 complementation groups. Analysis of the content of cytochrome c oxidase subunits in each mutant permitted the definition of three phenotypic classes. One class contains three complementation groups whose strains carry mutations in the COX4, COX5a, or COX9 genes. These genes encode subunits IV, Va, and VIIa of cytochrome c oxidase, respectively. Mutations in each of these structural genes appear to affect the levels of the other eight subunits, albeit in different ways. A second class contains nuclear mutants that are defective in synthesis of a specific mitochondrial-encoded cytochrome c oxidase subunit (I, II, or III) or in both cytochrome c oxidase subunit I and apocytochrome b. These mutants fall into 17 complementation groups. The third class is represented by mutants in 14 complementation groups. These strains contain near normal amounts of all cytochrome c oxidase subunits examined and therefore are likely to be defective at some step in holoenzyme assembly. The large number of complementation groups represented by the second and third phenotypic classes suggest that both the expression of the structural genes encoding the nine polypeptide subunits of cytochrome c oxidase and the assembly of these subunits into a functional holoenzyme require the products of many nuclear genes.     

The characterization and role of zinc binding in yeast Cox4

Yeast Cox4 is a zinc binding subunit of cytochrome c oxidase. Cox4 is the only cofactor-containing subunit that is not directly part of the catalytic core of the enzyme located in the mitochondrial inner membrane. The Zn(II) site is shown to be distinct from the bovine ortholog, as it results from the x-ray structure of the entire cytochrome c oxidase in having a single histidyl residue and three conserved cysteines residues in the coordination sphere. Substitutions at the Cys ligand positions result in non-functional Cox4 proteins that fail to lead to cytochrome oxidase assembly. Limited function exists in His-119 mutants when overexpressed. Zn(II) binding in Cox4 is, therefore, important for the stability of the complex. The solution structure of yeast Cox4 elucidated by multidimensional NMR reveals a C-terminal globular domain consisting of two beta sheets analogous to the bovine ortholog except the loop containing the coordinating His in the yeast protein and the fourth Cys in the bovine protein are in different positions in the two structures. The conformation of this loop is dictated by the different sequence position of the fourth coordinating zinc ligand. The Zn(II) ion is buried within the domain, consistent with its role in structural stability. Potential functions of this matrix-facing subunit are discussed.     

Mitochondrial targeting signal of rat liver monoamine oxidase B is located at its carboxy terminus.

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 55 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 b5. 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.