Comparison of the Primary Structures of Cytochrornes c from Wild and Respiration-deficient Mutant Yeasts

Transcription

1 The Journal of Biochemistry, Vol. 61, No. 1, 1967 Comparison of Primary Structures of Cytochrornes c from Wild and Respiration-deficient Mutant Yeasts By YOSHIHITO YAOI* (From Institute for Protein Research, Osaka University, Osaka) (Received for publication, June 24, 1966) The primary structures of cytochromes c from Saccharomyces oviformis, S. cerevisiae and ir respiration-deficient mutants were com pared by criteria of chromatograms on Amberlite CG-50 columns, amino acid compositions and peptide maps of tryptic digests. It was concluded that se strains contained a cytochrome c of same primary structure. A well known respiration-deficient mutant (RD mutant) of yeast, which can be obtained by acriflavin treatment of wild type, exhibits interesting biochemical and genetic properties (1, 2 ). The respiration deficiency in such a vegetative (cytoplasmic) RD mutant is known to be inherited in an extrachromo somal manner (1). This mutant almost lacks cytochromes a and b under usual conditions** (1, 2), but contains nearly normal amount of cytochrome c (1-4 ). At first sight, it is a curious fact that only cytochrome c remains in this mutant, whereas cytochromes a and b are deficient. In present investigation, such an RD mutant was isolated from Saccharomyces ovifo rmis M-2, and primary structure of its cytochrome c was compared with that from wild type, of which amino acid sequence had been previously determined in our labo ratory (5-12). Moreover, primary structure of cyto chrome c from a standard strain of S. cerevisiae was compared with that from S. oviformis M-2. Since S. cerevisiae has widely been investigated from biochemical and genetic view points, it seems to be of value to know primary * Present address: Division of Biology Cancer Center Research Institute, Tokyo. personal, National ** Tagawa, K., Ishidate, K., and Hagihara, B., communication. 54 structure of S. cerevisiae cytochrome c when one would like to consider primary struc ture in relation to physiological or ge netic problems. Cytochrome c from RD mutant of S. cerevisiae was also preliminarily investigated. EXPERIMENTAL Strains JY-1 (Saccharomyces cerevisiae, wild type, haploid cell, mating type a) was supplied from stocks of Dr. C.C. Lindegren, Carbondale, U.S.A., by Dr. S. Nagai, Nara Women's University, Nara. It corresponds to Nagai's strain No. 12. S. oviformis M-2 was strain stocked in Sankyo Co., Ltd., Tokyo. Isolation of RD Mutant-The wild type yeast (S. oviformis M-2) was inoculated in 5 ml. of medium containing 1 % Bacto yeast extract, 1 % Bacto peptone, 2% glucose and % acriflavin. It was incubated at 30 C for two days. The RD mutant was selected referring to its small colony sizes and inability to grow on a plate containing 3% glycerol as a sole carbon source. One typical RD mutant was thus selected and designated as MRD-1. Only cytochrome c band at 550 mĐ was detected spectroscopically in MRD-1. This strain was used as a cytoplasmic RD mutant, though genetic analysis was not performed. Cultures-For large scale cultivation of JY-1, 7 liters of seed culture was inoculated in 650 liters of medium containing 1% Daigo yeast extract, 1% Daigo peptone and 2% glucose, and incubated at 30 C with air-bubbling. Fifteen kg. of wet cells could be harvested from 40-hour culture. The large scale cultivation of MRD-l was kindly performed

2 Cyt. c from RD Mutant of Yeast 55 in Tanashi plant of Sankyo Co., Ltd., Tokyo, at 30 C for 20 hours. In small scale experiments, MRD-1 was culti vated in above medium at 30 C for 40 hours with shaking, and 25 g. of wet cells were harvested from 2.5 liters of culture. Isolation of Cytochrome c-cytochromes c from JY-1 and small scale culture of MRD-1 were extracted and purified according to method of Hag i h a r a el al. (13 ). Cytochrome c from large scale culture of MRD-1 was extracted in laboratory of Sankyo Co., Ltd., and purified in this laboratory by chromatography on an Amberlite CG-50 column. The S. oviform is cytochrome c used here is that previously used for studies on amino acid sequence (5-12). Amino Acid Analysis-About 3 mg. of each cyto chrome was hydrolyzed with 6 N HCl at 110 C in vacuo for 20 hours, and amino acid composition was determined by a Beckman/Spinco automatic amino acid analyzer. Peptide Map of Tryptic Digest-Each cytochrome c (10 mg.) was dissolved in I ml. of 1 % (NH4)HCO3, ph 9.0. To this solution was added 0.1 mg. of trypsin [EC ] (Worthington Biochemical Corp.) and mixture was incubated at 37 C for 4 hours. The lyophilized digest was again dissolved in a small amount of deionized water, and an aliquot (corresponding to 2 mg. protein) was placed on a sheet of a Toyo filter paper No. 51 (60 X 60 cm.). Peptides were first separated by paper chromatography (n-butanol-pyridine-acetic acid-water, 15:10:3:12, v/v) and n by paper electrophoresis (ph 3.6, 3,000 v/60 cm., 70 minutes) in second dimension (61). The detection of peptides was performed by ninhydrin, Pauly and Ehrlich reagents. FIG. 1. Chromatogram of MRD-1 cyto chrome c (oxidized form) on a column of Amberlite CG-50. The extract from 25 g. of MRD-1 cells (wet weight) was loaded on a column of Amberlite CG-50 ( mesh, 1 X 7 cm.) which was pre viously equilibrated with 0.25 M ammonium phos phate buffer of ph 7.0. Elution was carried out with 0.35 M and 2.0 M ammonium phosphate buffers of ph 7.0 as indicated in figure. TABLE I Amino Acid Compositions of Cytochromes c from Different Strains of Yeast1) RESULTS Chromatography of Cytochrome c-a typical chromatogram of cytochrome c from MRD-l on an Amberlite CG-50 column is shown in Fig. 1. The main component was fraction I. Fraction II, which was eluted with 2 M am monium phosphate buffer, was composed of dimer molecule of fraction I (14,15). This pattern is identical with that of S. oviformis M-2 cytechrome c (5). Calculating from absorbancy at 408 mt ( molecular extinction coefficient of oxidized cyto chrome c at 408 mp was assumed to be 10.7 x 104) (16), 1.4 mg. of cytochrome c, including dimer molecule, was obtained by 1) Values were obtained in a single analysis with 20 hours' acid hydrolyzate and presented in terms of molar ratios. Tryptophan and half cystine were not determined. 2) Theoretical values (5 ).

3 56 Y. YAOI FIG. 2. Peptide maps of tryptic digests of cytochromes c from different strains of yeast. Two mg. of digests were first separated by paper chromatography (n-butanol-pyridine-acetic acid water, 15:10:3: 12, v/v), and n by paper electrophoresis at ph 3.6 and 3,000 v/60 cm. for 70 minutes, in 2nd dimension. Peptides showing initial yellow ninhydrin color are indicated by symbol Y. Symbols P and E indicate that peptides were positive to Pauly and Ehrlich reagents, respecti vely. chromatography from 25 g. of wet cells of MRD-1. This value seems to be consistent with cytochrome c content of S. oviformis M-2 (about 0.001% of wet cells) (17), if correction is made for loss occurring during process of concentration and column chromatography of cytochrome. The chromatogram of cytochrome c from JY-1 was also same as in two cases mentioned above. Moreover, main com ponent (fraction I) obtained from each of se strains, MRD-1 and JY-1, behaved as a single band on a column of Amberlite CG - 50, when it was loaded on column in a mixture with M-2 cytochrome c. The fol lowing studies on amino acid composi tions and peptide maps were performed with fractions I obtained from se strains. Amino Acid Compositions-As can be seen in Table I, amino acid compositions of cytochromes c from MRD-1 and JY-1 agreed with that of S. oviformis M-2. Besides values listed, M-2 cytochrome c contains one mole of tryptophan and three moles of half-cystines (5). Although y were not analyzed quantitatively for cytochromes c from MRD-1 and JY-1, se contents were suggested to be also identical with those in M-2 cytochrome c, by following peptide map experiments. Peptide Maps-As shown in Fig. 2, peptide maps of tryptic digests of cytochromes c obtained from three strains are essentially same. Slight differences observed in se peptide maps could also be seen in maps of different tryptic digests of same preparation of M-2 cytochrome c. Since amino acid sequences of peptides extracted from major spots on map of cytochrome c have been previously established (5,8-12), it could be concluded that peptides essential to assess total amino acid sequence of cyto chrome c were mutually identical in se three samples of cytochrome c. DISCUSSION From genetic plasmic respiration-deficient is considered to contain point of view, cyto mutant of yeast wild type cyto-

4 Cyt. c from RD Mutant of Yeast 57 chrome c, since structural gene for cytochrome c seems not to be affected in this mutant (1, 4,18 ). However, Slonimski et al. (19) have recently reported that wild type yeast contained two isocytochromes c (iso -1 and iso-2), which could easily be separated on a column of Amberlite CG-50, and proportion of two isozymes varied during growth of cells. Since cytochrome content in yeast cells is known to be greatly dependent on partial pressure of oxygen and concentration of catabolites in media, such as glucose (20, 21), propor tion of isocytochromes in RD mutant, which does not respire, may be different from that of wild type. In present experiments, however, no "iso-2" type cytochrome c was detected in JY-1 and MRD-1. They contain only "iso-1" type cytochrome c which was shown to have same primary structure as described above. The "iso-2" type cytochrome c could not be detected in present experiments, probably because its content was so low as was reported thus far (about 4% of total cytochromes c) (4). On or hand, cytochrome c from a cytoplasmic RD mutant of S. cerevisiae JY-1 was also preliminarily examined. This mutant was demonstrated to be cytoplasmic mutant in its genetic and biochemical pro perties and designated as JY-10*. It also contained "iso-1 " type cytochrome c, of which amino acid composition and peptide map of tryptic digest were also shown to be similar to those of wild type. These data seem to support assump tion that cytoplasmic mutation does not affect genetic systems which determine both amino acid sequence of cytochrome c at least of " iso-l " type, and propor tion of isocytochrome c in yeast. Recently, it was reported by H e l l e r and Smith (22) that poky mutant of Neurospora crassa, which corresponded to RD mutant of yeast in its genetic and biochemical properties, also contained wild type cytochrome c. * Yaoi and Okazaki, manuscript in preparation. This is consistent with present results obtained with yeast mutant. The author is grateful to Sankyo Co., Ltd., Tokyo, and Dr. M. Shirasaka for kind co-operation with cultivation of yeasts and extraction of cytochromes. He also thanks Prof. K. Narita and Prof. K. Okunuki for guidance and encourage ment during study. Thanks are also due to Miss F. Hata for performance of amino acid analyses with an automatic equipment. REFERENCES (1) Ephrussi, B., " Nucleo-cytoplasmic Relations in Micro organisms", Clarendon Press, Oxford, p. 15 (1953) (2) Sherman, F., and Slonimski, P.P., Biochim. et Biophys. Acta, 90, 1 (1964) (3) Mackler, B., Douglas, H.C., Will, S., Hawthorne, D.C., and Mahler, H.R., Biochemistry, 4, 2016 (1965) (4) Sherman, F., Taber, H., and Campbell, W., J. Mol. Biol., 13, 21 (1965) (5) Narita, K., Titani, K., Yaoi, Y., and Murakami, H., Biochim. et Biophys. Acta, 77, 688 (1963) (6) Narita, K., Murakami, H., and Titani, K., J. Biochem., 56, 216 (1964) (7) Yaoi, Y., Titani, K., and Narita, K., J. Biochem., 56, 222 (1964) (8) Titani, K., Kimura, M., Vanecek, J., Murakami, H., and Narita, K., J. Biochem., 56, 230 (1964) (9) Titani, K., and Narita, K., J. Biochem., 56, 241 (1964) (10) Narita, K., and Titani, K., J. Biochem., 56, 257 (1964) (11) Yaoi, Y., J. Biochem., 59, 236 (1966) (12) Yaoi, Y., Titani, K., and Narita, K., J. Biochem., 59, 247 (1966) (13) Hagihara, B., Horio, T., Yamashita, J., Nozaki, M., and Okunuki, K., Nature, 178, 629 (1956) (14) Motonaga, K., Misaka, E., Nakajima, E., Ueda, S., and Nakanishi, K., J. Biochem., 57, 22 (1965) (15) Motonaga, K., Katano, H., and Nakanishi, K., J. Biochem., 57, 29 (1965) (16) Margoliash, E., Brit. med. Bull., 9, 89 (1963) (17) Nakanishi, K., Katano, H., Motonaga, K., Ito, T., and Haga, M., J. Japan. Biochem. Soc., (in Japanese), 36, 102 (1964) (18) Sherman, F., Genetics, 49, 39 (1964) (19) Slonimski, P.P., Archer, R., Pere, G., Sels, A., and Somlo, M., " Mechanismes de Regulation des Activitis Cellulaires chez les Microorganismes", Edi tions du Centre National du la Recherche Scienti fique, Paris, p. 435 (1965)

5 58 Y. YAOI (20) Ephrussi, B., and Slonimski, P.P., Biochim. et Biophys. Acta, 6, 256 (1951) (21) Polakis, E.S., Bartley, W., and Meek, G.A., Biochem. J., 90, 363 (1964) (22) Heller, J., and Smith, E.L., Proc. Natd. Acad. Sci. U.S., 54, 1621 (1964)