J. Biochem. 103, 954-961 (1988)

Construction of a Human Cytochrome c and Its Functional Expression in

Yoshikazu Tanaka,* Toshihiko Ashikari,* Yuji Shibano,* Teruo Amachi,* Hajime Yoshizumi,* and Hiroshi Matsubara** *Institute for Fundamental Research, Research Center, Suntory, Ltd., Shimamoto-chou, Mishima-gun, Osaka 618; and **Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 530

Received for publication, December 14, 1987

The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.

Cytochrome c is a c-containing found in the Ltd. mitochondria of all eukaryotic cells and functions as an Recombinant DNA Technique-Oligonucleotides were esssential component of the energy-yielding mitochondrial chemically synthesized by a DNA synthesizer (Applied electron transfer system. It is a small protein, easily Biosystems, 381A). Conventional recombinant DNA tech isolated, and is one of the best known as regards niques were previously described by Maniatis et al. (15). structure-function and evolutionary relationships (1). Site-specific mutagenesis was performed by the methods of sequences of cytochromes c are known for Zollar and Smith (16) and Morinaga et al. (17). scores of species (1). On the other hand, to our knowledge, Construction and Screening of cDNA Library-A cDNA complete nucleotide sequences of functional cytochrome c prepared from a mixture of human embryo and placenta by are available for only six species; S. cerevisiae (2, 3), using the cDNA synthesis kit was cloned into the Pstl site Schizosaccharomyces pombe (4), of pBR322 by dG/dC tailing (18). The constructed cDNA (5), chicken (6), rat (7), and mouse (8). Nucleotide library consisted of 400,000 clones. sequences of some pseudogenes from rat (9, 10), mouse Construction of Human Genomic Library-A genomic (8), and human (11) are also available. The amino acid library was constructed by partially digesting genomic sequence of human cytochrome c is known (12) but that of DNA from a human placenta with the restriction endonu human functional cytochrome c gene is not. clease EcoRI and ligating fragments approximately 16-20 Cytochrome c is known to protect the ischemic myocar kilobases (kb) long onto the purified EcoRI arms of the dium during acute coronary occlusion (13) ; bovine and lambda phage Charon 4A (15). horse cytochromes c are used clinically. It is, however, Yeast Strains and Plasmids-The S. cerevisiae strains practically impossible to obtain human cytochrome c from used in this work were XS-30-2B (MATa, his3, ura3, leu2, human organs. It may be possible to obtain human cyto trpl) (19) and XS-30-2B1CYC1 obtained in this experi chrome c on a large scale from a recombinant microorga ment, which was a Leu+ transformant resulting from nism such as S. cerevisiae. integration of the LEU2 gene into the above strain. The

During our research, Scarpulla and Nye reported that rat plasmid pYGA2269 (20) is a shuttle vector which contains cytochrome c was functionally expressed in yeast, which a glyceraldehyde-3-phosphate dehydrogenase (GAP) pro can serve as a host organism for the efficient functional moter, an inverted repeat and an autonomously replicating expression of a mammalian cytochrome c (14). sequence from 2 ,ƒÊm plasmid, TRP1, as a selectable Here, we report the nucleotide sequences of partial marker. The coding sequence to be expressed can be cDNA and new three pseudogenes of human cytochrome c inserted just after the GAP promoter by use of its EcoRI and the construction of human cytochrome c gene function and SalI sites (Fig. 1). The plasmid pYGA2269 was ally expressed in yeast. digested by EcoRI, repaired and ligated with BamHI linker. The constructed plasmid was denoted as pYGA2269EB. EXPERIMENTAL PROCEDURES Construction of Human Cytochrome c Gene-Human cytochrome c gene was constructed on the basis of Materials-Conventional enzymes for recombinant DNA pHGC3K5, which was obtained in this experiment and techniques were obtained from Toyobo Co., Ltd. and encoded five displaced amino acid residues compared with Takara Shuzo Co., Ltd. The cDNA kit, [ƒ¿-32P]dCTP and the authentic human cytochrome c amino acid sequence, [ ƒÁ-32P] ATP were the product of Amersham Japan, Ltd. formed by changing the codons in vitro as follows. EcoRI The DNA sequence kit was purchased from Toyobo Co., and Sail sites were also made just before the initial codon ATG and just after the stop codon, respectively. Six Abbreviations: GAP, glyceraldehyde-3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; SOD, superoxide dismutase; kb, oligonucleotides were synthesized to perform the desired kilobase; bp, base pairs; N-, amino mutations, as shown in Table I.

954 J. Biochem. Functional Expression of Human Cytochrome c in Yeast 955

TABLE I. Sequences of oligonucleotides for in vitro mutagenesis. *Mutated base.

Fig. 1. The restriction maps of pYGA2269, pYHCC101, and pYHCC105. The solid boxes denote the coding regions of human cytochrome c genes. IR, inverted repeat of 2 ,ƒÊm plasmid; Ap, ampicilin resistance; ARS, automonously replicating sequence; PGAP, promoter of glyceraldehyde-3.phosphate dehydrogenase; CEN4, centromere 4; TRP1, TRP1 gene; URA3, URA3 gene; E, EcoRI; H, HindlIl; S, Sail; X, Xhol.

An about 0.5 kb EcoRI fragment of pHGC3K5 was inserted into the EcoRl site of M13mp19. Between the two thus constructed were denoted as pYHCC101 and possible orientations, the bacteriophage M13-K5E1, whose pYHCC110, respectively. On the plasmids pYHCC101 and single strand DNA could hybridize the oligomers, was pYHCC110, human cytochrome c genes were regulated by selected. The oligomer A098 was used to introduce the GAP promoter, which is known to be a strong promoter in site-specific change in the single strand form of M13-K5E1 yeast (20). They are YEp type plasmids and have TRPI as (16). After completion of the procedure and detection of the selectable marker (Fig. 1). The plasmid pYHCC102 the altered M13 phage by plaque hybridization with the which has phosphoglycerate kinase (PGK) promoter in labeled oligonucleotide, and alteration was confirmed by stead of GAP promoter of pHCC101 was also constructed in DNA sequencing. The desired phage was denoted as the same manner. M13-K5E1-1. Next, the oligomer A094 was used to An about 1.7 kb Hind‡V fragment of pYHCC101 was introduce site-specific change in the single strand of inserted into one of the Hind‡V sites of a YCp type plasmid M13-K5E1-1. After repeated mutagenesis by A098, A094, YCp19. The constructed plasmid was denoted as A096, A097, and A109 in the single-strand DNA of pYHCC105 (Fig. 1). M13-K5E1, M13-K5E1-5 was obtained. Construction of the Plasmid Containing CYC1 Gene Mutation by A095 was introduced as follows, since -CYC1 gene including both flanking regions had been mutation by A095 was not achieved by the same method. obtained from genomic DNA of XS-30-2B in our laboratory The 0.3 kb EcoRI-SalI fragment from the double-strand following the restriction map of Smith et al. (2). The 0.6 DNA from M13-K5E1 was inserted into the plasmid pUC9. kb TagI fragment of CYC1 gene (2), which contains 0.3 kb The site-specific mutagenesis by the oligomer A095 was of 5•Œ-non-coding region, the whole coding region and 20 by performed by the method of Morinaga et al. (17) with the of 3•Œ-non-coding region, was inserted into the AccI site of use of the 2.6 kb EcoRI-Hind‡V fragment and the 2.6 kb pUC9. Of two possible orientations, pUC-CYC1 whose Dral fragment of the inserted pUC9. The plasmid finally BamHI-SalI fragment was 0.6 kb long was selected. constructed was denoted as pHCC110. BamHI-digested pUC-CYC1 was degraded by an exonu Another way used to construct the cytochrome c gene was clease Bal3l by the method of Maniatis et al. (15) and as follows. A 0.26 kb Bgl‡U-PstI fragment of pHCC71 was ligated with a BamHl linker. The plasmid pUC-CYCl-227 subcloned into BamHI-PstI sites of M13mp18. A Sail site whose BamHI linker was inserted at 65 by upstream from was made by A134 (Table I) on the subcloned template. the initial codon ATG of CYC1 was subjected to the The desired phage was denoted as M13-HCC71-S. The following studies. 80-base-pair (bp) Sau3AI-SalI fragment from the double The plasmid pYCYCl-227 was obtained by ligating 0.4 stranded DNA of M13-HCC71-S and the 0.25 kb EcoRI- kb BamHI-SalI fragment of pUC-CYC1-227 with 8 kb Sau3AI fragment from pGCC110 were ligated with EcoRI- BamHI-SalI fragment of pYGA2269EB. An about 1.8 kb SaII digest of pUC9. The constructed plasmid was denoted Hind‡V fragment of pYCYCl-227 was inserted into one of as pHCC101. the Hind‡V sites of YCp19 and the inserted plasmid was Successful construction of human cytochrome c genes denoted as pYCYCl-227-C. was confirmed by sequence studies of the coding regions of Media for Yeast-Yeast strains were grown at 30•Ž in pHCC101 and pHCC110. The plasmids pHCC110 and minimum media (0.67% yeast nitrogen base without amino pHCC101 encode the same amino acid sequences but have acids (Difco), 0.05% yeast extract (Difco), and necessary one base difference in the coding regions; the codon of supplements) containing 1% glucose (YNED), 1% DL- Leu-94 is TTG in pHCC110 but TTA in pHCC101. This is sodium-lactate (YNEL) (14), or both 1% glucose and 1% because the coding region of pHCC101 is partly derived DL-sodium lactate (YNEDL), or in Burkholder's medium from the cDNA. (21). Introduction of Human Cytochrome c Genes into Yeast Insertional Disruption of CYC1-The 0.6 by EcoRI Expression Vector-Each of the 0.3 kb EcoRI-SalI frag - Hind‡V fragment of CYC1 gene, which encompassed most ments from pHCC101 and pHCC110 was ligated with 8 kb of the coding region and the 3‡V-non-coding region, was EcoRI-SalI fragment of pYGA2269 (20). The plasmids inserted into pUC18 (pUGC3EH). The inserted plasmid

Vol. 103, No. 6, 1988 956 Y. Tanaka et al.

Fig. 3. Comparison of restriction maps of sequenced regions of

pHCC71, pHGC3K5, pHGC4E1, and pHGC14E4. The initial codon ATG and the stop codon TAA are shown by arrows. Coding regions are shown by thick lines. pHGC14 contains an insertion in its coding region. E, EcoRI; G, BglII; H, Hind‡V; P, PstI; U, Sau3AI.

cultured cells grown at 30•Ž in YNED medium was inoculated into 50 ml of YNEL medium and cell growth was monitored in terms of absorbance at 660 nm. Preparation of the Recombinant Cytochrome c from Yeast-The strain XS-30-2B CYC1 containing pHCC101 was cultivated at 30•Ž in Burkholder medium (21) contain ing 2% casamino acid. Instead of glucose, 2% DL-lactate was used as a sole carbon source. About 50 g of cells were obtained from a 7 liter broth. The yeast cells suspended in 50 ml of 1 M NaCl and 25 ml of ethyl acetate were shaken gently for about 18 h (23). The resulting autolysate was diluted with water and adsorbed on Amberlite CG-50 as described by Sherman et al. (23). Crude cytochrome c solution eluted from Amberlite CG-50 (23) was dialyzed against water and lyophilized. The lyophilized powder was resolved in a minimum volume of water and chromato graphed on a column (1 •~ 100 cm) of Sephadex G-75 equilibrated with 50 mM Tris-HCl, pH 7.5 and 150 mM NaCl. The eluate from the column was dialyzed against 10 mM sodium phosphate buffer, pH 7.2 and charged on a Fig. 2. Construction of a plasmid pUGC35B for disruption of column (0.5 •~ 10 cm) of CM-52 equilibrated with the CYCI in yeast. The open boxes and solid boxes denote the cyto chrome c and LEU2 genes, respectively. The details are given in the phosphate buffer. Cytochrome c was eluted with a linear text. A, AccI; B, BamHI; E, EcoRI; H, Hind‡V; K, KpnI; P, PstI; gradient of NaCl from 0 to 200 mM. The major cytochrome X, XhoI c fraction was subjected to further studies. Protein Analyses-Amino acid composition and amino (N)-terminal sequence were determined by an amino acid was digested by AccI, made blunt by using the Klenow analyzer (Hitachi 835) and a gas phase sequencer (Applied fragment, and ligated with Pstl linker (pUGCS3EH). At Biosystem 470A), respectively. Conventional protein this PstI site, the 4.0 kb PstI fragment of YEp13 was analyses were performed as previously described (24). inserted (pUGCS35). The partial digest of pUGCS35 by

EcoRI was repaired and BamHI linker was inserted RESULTS (pUGCS35B) (Fig. 2). The yeast XS-30-2B (Mates, trpl, ura3, his3, leu2) was transformed by the BamHI-Hind‡V Cloning of Human Cytochrome c cDNA-The 79mer digest of pUGCS35B and transformants were selected for nucleotide specific to human cytochrome c, which is the ( ) their ability to restore XS-30-2B to leucine prototrophy. strand of the nucleotide sequence coding for the carboxyl One of them was denoted as XS-30-2B•¢CYCl. The terminal 26 amino acid residues and the first base of stop method of yeast transformation was described by Ito et al. codons, was synthesized. Degenerate codons were arbitrar (22). ily selected. The sequence was as follows; 5•ŒACTCCTTG Cell Growth Analysis XS-30-2B CYC1 was trans GTGGCCTTCTTGAGGTAAGCGATGAGGTCGGCCCGCT formed by various plasmids and transformants were CCTCCTTCTTCTTGATGCCGACGAAGATCATCTT3•Œ. selected for their ability to restore XS-30-2B•¢CYCl to The human cDNA library was screened by a colony tryptophan prototrophy. Individual isolates containing the hybridization method (15) , using the 32P-labeled 79mer plasmids were then tested for growth on DL-lactate as a sole probe. Two positive clones were obtained. Their plasmids carbon source at 30•Ž and compared to the wild-type control were the same and were denoted as pHCC71. The insert on strain expressing normal levels of yeast cytochrome c. pHCC71 was subjected to sequence study and the results For a quantitative comparison of cell growth, 0.5 ml of are shown in Fig. 1S. The sequence study revealed that it

J. Biochem. Functional Expression of Human Cytochrome c in Yeast 957

Fig. 4. Comparison of nucleotide sequences of coding regions. (a), pHGC3; (b), pHGC14; (c), pHGC4; (d), H201 (11); (e), H202 Fig. 5. Comparison of nucleotide sequences of 3•Œnon-coding (11); (f), RC4 (7); (g), pHCC71. The complete sequence of the regions. (a), pHCC71; (b), pHGC3; (c), pHGC14; (d), pHGC4; (e), coding region of pHGC3 is presented and only those nucleotides that RC4. The complete sequence of pHCC71 is presented and only those differ from pHGC3 are shown for the other sequences. The number nucleotides that differ from pHCC71 are shown for the other ing system begins from the initial codon. Deleted nucleotides are sequences. The nucleotide sequences are aligned for maximum shown by -. The inserted sequence of pHGC14 is omitted. The homology. The numbering system begins from the stop codon. A beginning of pHCC71 is shown by an arrow. period in each sequence indicates the end of the sequence.

contained a 0.1 kb coding region and a 1 kb 3•Œ-non-coding kb Bgl‡U fragment bf pHCC71, respectively. The restric region of human cytochrome c cDNA (Fig. 3). The coding tion map of the sequenced region of pHGC3K5 is shown in region encoded the carboxyl-terminal 28 amino acid resi Fig. 3. It encoded cytochrome c-like protein. dues of the published human cytochrome c amino acid The 50 positive plaques mentioned above were screened sequence (12). A 17mer oligonucleotide was synthesized with nick-translated 0.5 kb EcoRI fragment of pHGC3K5. on the basis of the cDNA sequence for further study. The Two clearly positive plaques were selected and their sequence was 5•ŒTTACTCATTAGTAGCTT3•Œ. recombinant lambda DNAs were denoted as pHGC4 and Cloning and Analyses of Three Pseudogenes of Human pHGC14. The 1.3 kb EcoRI fragment of pHGC4 and 2.4 kb Cytochrome c-Since we failed to obtain the full length of EcoRI fragment of pHGC14, both of which hybridized with cDNA, we screened the human genomic library by plaque 0.5 kb EcoRI fragment of pHGC3K5, were each subcloned hybridization using the nick-translated 0.6 by Bgl‡U frag into the EcoRI site of pUC9 (denoted as pHGC4El and ment of pHCC71. Among approximately 1,000,000 pHGC14E4, respectively). The restriction maps of the sequenced regions of plaques, 50 positive plaques were obtained. One of them, which contained the recombinant lambda DNA denoted as pHGC4El and pHGC14E4 are shown in Fig. 3. The pHGC3, was further studied. sequences of 0.8 kb EcoRI-Hindlll fragment of pHGC4E1 A southern hybridization experiment revealed that the 6 and 1.2 kb Bgl‡U fragment of pHGC14E4 are shown in Figs. kb KpnI fragment of pHGC3 hybridized with the 79mer 3S and 4S, respectively. Both contained nucleotide se probe. The KpnI fragment with inserted pUC18 was quences homologous to the cytochrome c gene. The denoted as pHGC3K5. The 0.5 and 0.6 kb EcoRI fragments nucleotide sequences of coding regions of pHGC3K5, of pHGC3K5 hybridized with the 17mer probe and the 0.6 pHGC4El, pHGC14E4, and pHCC71 are compared in Fig.

Vol. 103, No. 6, 1988 958 Y. Tanaka et al.

Fig. 7. Absorption spectra of reduced cytochromes c in the autolysates with ethyl acetate. Cells (0.6 g) cultured on YNEDL medium (0.67% yeast nitrogen base, 0.05% yeast extract, 1% glucose, 1% DL-lactate, and necessary supplements) were suspended in 1 ml of NaCl and 0.5 ml of ethyl acetate and the autolysates were obtained. The autolysates reduced by sodium dithionite were subjected to Fig. 6. Growth of the transformants and the host in YNEL spectral measurements. A, XS-30-2B; B, XS-30-2B•¢CYCl. medium which contains 0.67% yeast nitrogen base without amino acid (Difco), 0.05% yeast extract, 1% DL-lactate, and necessary supplements. Cell growths were measured as the

absorbance at 660 nm. A, XS-30.2B; B, XS-30-2B (pYCYCl-227 CYCl. C); C, XS-30-2B•¢(pYCYC1.227); D, XS-30-2B•¢CYC1(pYHCC Characterization of the Recombinant Human Cyto 101); E, XS-30-2BJCYC1(pYHCC110); F, XS-30-2BJCYC1 chrome c-The amino acid composition of the recombinant (pYHCC105); G, XS-30-2B CYC1(pYHCC102); H, XS-30.2B•¢ CYC1. cytochrome c agreed with that of the authentic cytochrome c (Table IS). Small amounts of e-N-trimethyl lysine and ƒÃ-N-monomethyl lysine were detected. The N-terminal 4. Reported sequences of H201, H202, and RC4 (4, 11) are amino acid sequence of the recombinant cytochrome c was also listed. pHGC3K5 encodes a cytochrome c-like protein determined to the 18th residue. It was Gly-Asp-Val-Glu which has five displaced amino acid residues compared with - Lys-Gly-Lys-Lys-Ile-Phe-Ile Met-Lys-X Ser-Gln-X-His the published human cytochrome c sequence. A one-base (X, no residue was detected). This sequence is consistent silent mutation was observed between the cDNA and with that of the authentic cytochrome c. Since heme c binds pHGC3K5. Within a coding region-like sequence, to the 14th and 17th cysteine residues, they could not be pHGC4E1 had one base deletion and 18 displaced amino directly identified. The isoelectric point of the recombinant acid residues compared with human cytochrome c. cytochrome c was more basic than that of horse cytochrome pHGC14E4 had a 316 by insertion and 8 displaced residues c. in the putative coding region. The 3•Œ-non-coding region of

pHCC71, pHGC3K5, pHGC4E1, pHGC14E4, and RC4 are DISCUSSION compared in Fig. 5. Long obviously homologous regions were not found in their 5•Œ-non-coding regions. Mammalian genomes are known to contain families of Functional Expression of Human Cytochrome c Gene in about 30 different sequences that are homologous to DNA Yeast-The growth curves of various yeast transformants encoding the somatic form of cytochrome c. This is true for and hosts are compared in Fig. 6. The constructed the , and some pseudogenes of human XS-30-2B•¢CYCl did not grow on lactate as a sole carbon cytochrome c are known (11). The three related sequences source (Fig. 6G). Its autolysate by ethyl acetate contained obtained here from the genomic library are supposed to be only a small amount of cytochrome c, which was assumed to pseudogenes for following reasons: 1) their sequences are be iso-2-cytochrome c encoded by CYC7 (Fig. 7). These different from the cDNA sequence and they can not code the results indicted that CYC1 gene was successfully disrupted reported human cytochrome c, 2) they have mutational in XS-30-2B•¢CYCl. The deficiency of CYC1 was naturally defects in the cytochrome c coding region including replace complemented by the plasmids carrying CYC1, or ments in codons for essential residues (10), 3) they do not pYCY1-227 and pYCYCl-227-C (Fig. 6, B and C). Yeast contain introns which are assumed to be contained by transformants containing human cytochrome c genes such human cytochrome c gene (11), 4) putative coding regions as XS-30-2B•¢CYCl (pYHCC101), XS-30-2B•¢CYCl of pHGC4 and pHGC14 contain a deletion and an insertion, (pYHCC110), and XS-30-2B•¢CYCl (pYHCC105) simi respectively, and 5) the coding region of pHGC3 failed to larly grew on DL-lactate as a sole carbon source after long complement CYC1 deficiency (data not shown; the results lag phases (Fig. 6, D, E, and F). Though their doubling will be reported in the near future). The obtained cDNA times in the exponential phase were comparable to those of had a long 3•Œ-non-coding sequence. It is known that rat XS-30-2B, XS-30-2B?CYCl (pYCYCl-227), and XS-30 cytochrome c mRNA is up to 1.4 kb long (4). -2B•¢CYC1 (pYCYCl-227-C) (Fig. 6, A, B, and C), the final It is possible to discuss the phylogenetic relationships of growths of the transformants with human cytochrome c cytochrome c genes by counting the number of mutations genes were smaller than those of the transformants with which have accumulated in a homologous region. Numbers

J. Biochem. Functional Expression of Human Cytochrome c in Yeast 959

TABLE ‡U. Numbers of substitutions per nucleotide in coding regions (below diagonal) and in 3•Œ-non-coding regions (above diagonal). The numbers are calculated as substituted bases per compared bases . If comparedd nucleotides are less than 100 bp, the corresponding numbers are parenthesized.

of nucleotide substitutions per site (substituted bases/ does not seem to cause any difference as far as yeast compared bases) among various cytochrome c genes are transformant growth is concerned. summarized in Table ‡U. Deleted or inserted nucleotides We have shown that the recombinant and authentic were not counted. Table ‡U indicates that HGC3 and HGC4 human cytochromes c have an identical amino acid se seem to be closely related to the functional gene. As quence except for the amino-terminal acetyl group. Cyto regards HGC3, an over 600 by sequence was compared chromes c from many animals and plants are acetylated at with the cDNA. Assuming a substitution rate of 4.9 x 10-9 the N-terminus but those from insects and many micro substitution/site/year (11), HGC3 and the functional gene organisms are not (1). Human cytochrome c has an are calculated to have diverged about 7.5 million years ago. N-acetylated glycine residue and yeast iso-1 cytochrome c HGC4 seems to have emerged before HGC3 and HGC14 has a free N-terminal threonine residue. Hallewell et al. and just after the rat-human split. reported that recombinant human superoxide dismutase We examined the functional expression of human cyto (SOD) produced by yeast and authentic human SOD are chrome c in yeast and identified the recombinant cyto acetylated, while endogeneous yeast SOD is not (26). This chrome c. The yeast transformant with a plasmid contain indicates that yeast can acetylate the N-terminal residue of ing a human cytochrome c gene grew using human cyto some foreign proteins expressed in the cells. Thus, we chrome c instead of yeast iso-1 cytochrome c. Since expected that the recombinant human cytochrome c by mammalian cytochromes c react well with yeast cyto yeast might have the acetylated N-terminus. However, the chromes c peroxidase and yeast cytochromes c react well recombinant human cytochrome c produced by yeast with bovine cytochrome c oxidase (25), it is not surprising described here had a free glycine residue judging from the that human cytochrome c can substitute for yeast iso-1 N-terminal sequence analysis and a higher isoelectric point cytochrome c functions in the yeast electron transfer than that of horse cytochrome c, whose isoelectric point is system. However, it is curious that translated human supposed to be as same as that of authentic human cyto apo-cytochrome c can be converted to holo-cytochrome c in chrome con the basis of their amino acid compositions. The a heterogeneous organism, yeast. As Scarpulla and Nye fact that cytochrome c with a free N-terminus can function pointed out, yeast can serve as a host organism for the in the electron transfer system indicates that the N functional expression of a mammalian cytochrome c (14). -terminal acetylation is not essential for human cytochrome A longer lag phase was observed in a transformant c to function. More detailed studies on a molecular basis containing human cytochrome c gene by Scarpulla and Nye would be necessary to confirm the non-essentiality of the (14). N-terminal acetyl group in cytochrome c function in the There are two differences between our results and respiratory system. Scarpulla and Nye's results (14). One of them is that a It is known that Lys-77 of yeast iso-1 cytochrome c is transformant strain containing a human cytochrome c gene tri-methylated (1). Recombinant human cytochrome c on the one-copy plasmid pYHCC105 could grow on lactate contains a small amount of trimethyl lysine and monometh medium as well as one containing a multicopy plasmid, yl lysine. This may indicate the partial trimethylation of while the transformant strain containing a rat cytochrome Lys-72 of the recombinant cytochrome c which corresponds c gene on the one-copy plasmid could not (14). The other is to Lys-77 of yeast iso-1 cytochrome c. Dimethyl lysine that the yeast transformant containing a human gene grew may not be detected since its retention time is very close to to a lesser extent than the wild strain and the transformant that of lysine in our amino acid analysis system. containing CYC1 gene. Thus, human cytochrome c gene In any case, the cytochrome c biosynthetic process is seems to be able to complement the deficiency of iso-1 generally conserved both in yeast and human. Further cytochrome c to a lesser extent than native CYC1 gene. On results on mutated cytochrome c will be reported in the the other hand, a yeast transformant containing a rat gene near future. grew as well as a wild strain (14). Since our vectors, a host strain, a promoter and an origin of cytochrome c gene, are We thank Drs. T. Hase, K. Wada, and S. Wakabayashi for useful different from theirs (14), it is difficult to compare both discussions. results strictly. It is interesting that pYHCC102 which has a PGK REFERENCES promoter instead of a GAP promoter in a similar plasmid 1. Dayhoff, M.O. (1972) Atlas of Protein Sequence and Structure, could grow very slowly. There may be a compatibility Vol. 5, National Biomedical Research Foundation, Silver Spring between a cytochrome c gene and a promoter. The silent 2. Smith, M., Leung, D.W., Gilliam, S., Astell, C.R., & Hall, B.D. mutation in Leu-94 between pYHCC101 and pYHCC110 (1979) Cell 16, 753-761

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3. Montgomery, D.L., Leung, D.W., Smith, M., Shallit, P., Faye, 17. Morinaga, Y., Franceschini, T., Inoue, S., & Inoue, M. (1984) G., & Hall, B.D. (1980) Proc. Natl. Acad. Sci. U.S. 77, 541-545 Bio/Technology 2, 636-639 4. Russell, P.R. & Hall, B.D. (1982) Mol. Cell. Biol. 2,106-116 18. Gubler, U. & Hoffman, B.J. (1983) Gene 25, 263-269 5. Limbach, K.J. & Wu, R. (1975) Nucleic Acids Res. 13, 631-644 19. Ashikari, T., Nakamura, N., Tanaka, Y., Kiuchi, N., Shibano, Y., 6. Limbach, K.J. & Wu, R. (1983) Nucleic Acids Res. 11, 8931 Tanaka, T., Amachi, T., & Yoshizumi, H. (1986) Agric. Biol. 8950 Chem. 50, 957-964 7. Scarpulla, R.C., Agne, K.M., & Wu, R. (1981) J. Biol. Chem. 256, 20. Yoshizumi, H., Ashikari, T., Nakamura, N., Kunisaki, S., 6480-6486 Tanaka, Y., Kiuchi, N., & Shibano, Y. (1987) J. Jpn. Soc. Starch 8. Limbach, K.J. & Wu, R. (1985) Nucleic Acids Res. 13, 617-630 Sci. 34,148-154 9. Scarpulla, R.C. & Wu, R. (1983) Cell 32, 473-482 21. Burkholder, P.R. (1943) Am. J. Bot. 30, 206-210 10. Scarpulla, R.C. (1984) Mol. Cell. Biol. 4, 2279-2288 22. Ito, H., Fukuda, Y., Murata, K., & Kimura, A. (1983) J. 11. Wu, C.-I., Li, W.-H., Shen, J.J., Scarpulla, R.C., Limbach, K.J., Bacteriol. 153, 163-168 & Wu, R. (1986) J. Mol. Evol. 23, 61-75 23. Sherman, F., Stewart, J.W., Parker, J.H., Inhaber, E., & 12. Matsubara, H. & Smith, E.L. (1963) J. Biol. Chem. 228,2732 Shipton, N.A. (1968) J. Biol. Chem. 243, 5446-5456 - 2753 24. Tanaka, Y., Ashikari, T., Nakamura, N., Kiuchi, N., Shibano, Y., 13. Zalewski, A., Goldberg, S., Krol, R., & Maroko, P.R. (1987) Am Amachi, T., & Yoshizumi, H. (1986) Agric. Biol. Chem. 50,1737 Heart J. 113,124-129 -1742 14. Scarpulla, R.C. & Nye, S.H. (1986) Proc. Natl. Acad. Sci. U.S. 25. Yamanaka, T. (1975) J. Biochem. 77, 493-499 83,6352-6356 26. Hallewell, R.A., Mill, R., Tekamp-Olson, P., Blacher, R., Rosen 15. Maniatis, T., Fritsch, E.F., & Sambrook, J. (1982) Molecular berg, S., Otting, F., Masiarz, F.R., & Scandella, C.I. (1987) Bio/ Cloning, Cold Spring Harbor Laboratory, New York Technology 5, 363-366 16. Zoller, M.J. & Smith, M. (1982) Nucleic Acids Res. 10, 6487 - 6500

Supplemental Materials

Figure 2S. The nucleotide sequence of a part of pHCCJKS. The deduced amino acid sequence Is also shown. Five displaced residues in comparison with the authentic cytochrome c and the initial methioning residue are parenthesized.

J. Biochem. Functional Expression of Human Cytochrome c in Yeast 961

Figure 35. The nucleotide sequence of a part of pHCC4EI. The deduced amino acid sequence is also shown. The initial methionine residue and displaced residues are parenthesized.

Table IS. Comparison of amino acid compositions

Figure 4S. The nucleotide sequence of a part of pHGC14E4. The deduced amino acid sequence is also shown. The initial methionine residue and displaced residues are parenthesized.

TMLys, Trimethyl lysine; MMLys, Monomethyl lysine;

n. d., not determined.

Vol. 103, No. 6, 1988