Vol. 138, No. 3
JouRNAL oF BACrzIRoLOGY, June 1979, p. 863-0 0021-9193/79/060863/08$02.00/0
Yeast Cytochrome c-Specific Protem-Lysine Methyltransferase: Coordinate Regulation with Cytochrome c and Activities in cyc Mutantst HANS H. LIAOt AND FRED SHERMAN* Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Received for publication 17 January 1979
The cytochromes c of fungi and higher plants contain one or two residues of e-N-trimethyllysine, whose biological role is unknown. A cytochrome c-specific Sadenosylmethionine:protein-lysine methyltransferase (methylase) activity was shown to be present in extracts of the bakers' yeast Saccharomyces cerevisiae, and basic kinetic properties of this enzyme are described. The specific activity of the methylase was lower in extracts of cells grown under conditions of catabolite (glucose) repression or anaerobiosis where cytochrome c levels were low, compared with cells grown under derepressed conditions where cytochrome c levels were high. During anaerobic-to-aerobic adaptation, the methylase was induced in parallel with cytochrome c, thus suggestiDng that the syntheses of cytochrome c and cytochrome c methylase are coordinately regulated. None of the cyc strains surveyed (cycl, cyc2, cyc3, cyc4, cyc5, and cyc6) had diminished levels of methylase, although some of them were completely or almost completely deficient in cytochrome c.
Cytochrome c isolated from fungi and higher plants has been found to contain e-N-trimethyllysine (4). Cytochrome c from Neurospora crassa (4), both iso-cytochromes c from the bakers' yeast Saccharomyces cerevisiae (5), as well as cytochromes c from other fungi (2, 6, 16) have e-N-trimethyllysine confined to a single position corresponding to the position of amino acid 72 in vertebrate cytochrome c, whereas cytochromes c from wheat germ (4) and other higher plants (2, 6, 16) have two e-N-trimethyllysine residues confined to positions 72 and 86. In contrast, vertebrate cytochromes c do not contain any trimethyllysine residues. Protein methylation, which is widespread in nature but of unknown functional significance (2, 16), is a posttranslational event, with the methyl group derived from S-adenosylmethionine. In N. crassa, a precursor-product relationship has been established between the unmethylated and methylated cytochromes c (22); chromatographic separation of the cytochromes c from S. cerevisiae also revealed, in addition to the major iso-l-cytochrome c and minor iso-2cytochrome c methylated proteins, a small
amount of the unmethylated forms (11), which are presumably the precursors of the methylated cytochromes c. Furthermore, a cytochrome c-
specific S-adenosylmethionine:protein-lysine methyltransferase has been purified recently from N. crassa (8) and shown to restrict methylation to the lysine residue at position 72 of native horse heart cytochrome c, thus appearing to be the enzyme responsible for the modification of cytochrome c in vivo. The biological function of cytochrome c methylation is unknown. Measurement of the stabilities toward denaturants (17), circular dichroic spectra (13), and kinetic parameters in reactions with cytochrome c peroxidase, oxidase, and succinate oxidoreductase (18) showed only minor, if any, differences between the methylated and unmethylated yeast iso-1-cytochromes c. However, recent evidence suggests that trimethylated iso-1-cytochrome c does have a greater affinity for mitochondria than does the unmethylated form (19); although the in vivo significance of this finding is unclear, it suggested that
t Report UR-3490-1496 from the U. S. Department of Energy at the University of Rochester, Department of Radiation Biology and Biophysics. t Present address: Department of Bacteriology and Immunology, University of California, Berkeley, CA 94720.
853
the lack of such modification may express itself phenotypically as impaired cytochrome c function. Therefore, in the investigation reported here, cyc mutants of yeast, which are deficient in cytochrome c levels and activities (7, 23, 27, 28), were screened for cytochrome c methylase activity, with the goal of possibly identifying a
854
LIAO AND SHERMAN
methylase-deficient strain among the cyc mutants and demonstrating a role for the modification in vivo. (The term methylase refers to the enzyme activity that transfers methyl groups from S-adenosylmethionine to proteins.) In addition, we investigated whether the enzyme was subject to the same regulatory constraints as cytochrome c, i.e. repression of synthesis by glucose (10) and anaerobiosis (9) and derepression by nonfermentable carbon sources and aerobiosis. If the methylase were specific for cytochrome c and necessary for its function, it was expected that both of these proteins would be coordinately regulated. MATERIAIS AND METHODS Materials. S-Adenosyl-L-[methyl- 14C]methionine (58 to 59 mCi/mmol) was obtained from Amersham/ Searle, horse heart cytochrome c was obtained from Sigma Chemical Co., and Aquasol scintillation fluid was obtained from New England Nuclear Corp. Strains of S. cerevisiae. The strains examined in this study included the normal strains D311-3A and D273-1OB and cytochrome c-deficient mutants derived from these normal strains by using a spectroscopic scanning procedure (23), a benzidine staining procedure (28) and a chlorolactate enrichment procedure (27). Also examined were the cycl-1 CYC7-1 strain B4927, which functions normally, although it contains a high level of exclusively iso-2-cytochrome c, and cytochrome c-deficient mutants derived from this strain (7). Mutation at the structural gene cycl results in deficiencies in the amount or function of iso-icytochrome c; mutations at any of the loci from cyc2 through cyc6 result in various degrees of deficiency of both iso-l-cytochrome c and iso-2-cytochrome c. Growth of yeast. Small quantities of yeast sufficient for the measurement of methylase activity were obtained by the growth of each strain on the surface of a single plate containing 1% yeast extract (Difco), 2% peptone (Difco), 2% agar, and either 1% sucrose or 2% glucose. Yeast were streaked as thin lines across the surface of the plates, a growth condition that allows for derepression of the cytochromes (27). After 3 days of incubation at 30°C, the cells were scraped off the plate, washed twice with distilled water, and stored at -30°C. Larger amounts of cells were obtained by growth in 10-liter batches of medium containing 1% Ardamine Z yeast extract, 2% technicalgrade peptone (Difco), 0.2% (vol/vol) polypropylene glycol, 34 mg of penicillin per liter, 100 mg of streptomycin per liter, and either 2% (vol/vol) ethanol or 5% glucose. Cultures were grown in 14-liter Fermentation Design fermentors at 30°C with stirring at 500 rpm and with aeration of 600 liters of air per min. The cells were harvested at 4°C by centrifugation in a refrigerated Lourdes 30-R Clini-Fuge centrifuge for 6 min at 2,000 rpm, washed twice with water, and stored at -300C. Anarobic cultures were obtained by growth at 300C in 12 liters of a medium containing 1% yeast extract (Difco), 3% glucose, 0.1% KH2PO4, 0.12% (NH4)2SO4, 42 ml of a solution of 0.29% ergosterol and 28% (vol/
J. BACTrERIOL.
vol) Tween 80 in absolute ethanol (resulting in final concentrations of 10 mg/liter and 1 ml/liter, respectively) (3), 100 mg of streptomycin per liter, 34 mg of penicillin per liter, and 0.25% (vol/vol) polypropylene glycol. Each of the cultures was inoculated with a 500ml preculture grown with minimum aeration in a volumetric flask filled to the neck. The 12-liter cultures were maintained air-free by continuous flushing with N2 (Matheson prepurified N2, further scrubbed of 02 by a model 4002 Oxy-Trap [Alltech Associates]) at 80 ml/min for 6 h before inoculation and throughout growth; the cultures were agitated by stirring at 500 rpm. After 18 h, the cultures were chilled to 40C, transferred to chilled centrifuge bottles by displacement with N2, and centrifuged as described above. Cells were washed twice with cold deaerated water and either used immediately for the adaptation experiment described below or stored at -30°C for the subsequent preparation of crude extract. Respiratory adaptation. Adaptation of anaerobically grown yeast to air was performed by suspending cells to a concentration of 16 g (wet weight) per liter in 8 liters of 40 mM potassium phosphate (pH 7.4), 0.3% glucose, 1% (vol/vol) ethanol, and 0.2% (vol/ vol) polypropylene glycol. The culture was aerated at 28°C in a 14-liter Fermentation Design unit with 600 liters of air per min while being stirred at 500 rpm (20). Portious of the culture were obtained by siphoning 1.5 liters into centrifuge bottles containing ice and centrifuging in a cold Lourdes Clini-Fuge centrifuge as described above. The pelleted yeast cells were washed with cold water and recentrifuged in a Beckman JS-13 rotor for 5 min at 6,000 rpm; they were then either resuspended in water for the determination of oxygen uptake capacity and cytochrome c content or stored at -30°C for the eventual measurement of methylase activity. Preparation of crude extracts. Crude extracts of yeast were prepared by disruption with glass beads in a Braun type 2876 homogenizer. Ten-gram (wet weight) batches were suspended with 20 ml of 0.1 M Tris-hydrochloride buffer (pH 8.9) and shaken for a total of 90 s with 20 g of beads (diameter, 0.45 to 0.5 mm) in a Teflon bottle kept cool by a jet of CO2 gas. The extract was decanted from the beads and freed of cell debris by centrifuging twice at 4°C for 15 min in a Beckman JA-20 rotor spun at 15,000 rpm in a Beckman J-21B centrifuge. Efficiency of cell breakage was estimated by inspection under the microscope to be greater than 95%. For the preparation of extracts from small quantities of cells grown on solid media, an adapter for the Braun homogenizer was custom designed and constWcted in our facilities. The adapter accommodates up to eight Eppendorf 1.5-ml polypropylene micro test tubes with attached caps in two tiers and was constructed of aluminum to allow efficient cooling by CO2 gas. Screws holding the two tiers of racks together also served to prevent the adapter itself from rotating during shaking of the yeast suspension. Optimal breakage was achieved by suspending 0.4 g (wet weight) of yeast with 1 g of glass beads in sufficient 0.1 M Trishydrochloride buffer (pH 8.9) to fill the micro test tube and shaking the cell suspension for a total of 2.5 min. The methylase-containing supernatant from the
VOL. 138, 1979
CYTOCHROME c-SPECIFIC METHYLTRANSFERASE
crude extract was obtained after centrifuging twice at 40C in an Eppendorf 3200 centrifuge, the first spin being performed in the same test tube used for cell breakage. The efficiency of breakage in the micro test tubes was only about 50%; variations in the proportion of yeast to glass beads did not improve the extraction since a lower ratio of yeast to glass beads (0.2:1) yielded an extract too dilute for the methylase assay, whereas higher ratios (1:1 or 1.4:1) diminished the breakage efficiency still further. Assay for cytochrome c-specific methylase. The assay procedure was adapted from that described for the enzyme in extracts of N. crassa (15) but was simplified by using paper filter disks for the collection of trichloroacetic acid-precipitated protein (14). A final volume of 0.1 ml of each reaction mixture consisted of 0.1 M Tris-hydrochloride (pH 8.9), 0.225 ,Ci of Sadenosyl-L-[methyl-'4C]methionine (used without dilution of the commercial product), 0.23 mM horse heart cytochrome c, unless otherwise stated, and various amounts of crude extracts. The reactions were carried out at 37°C, usually for 15 min. Preincubation of the mixture for 5 min before the addition of Sadenosyl-L-[methyl-'4C]methionine had no effect on the subsequent incorporation of methyl-'4C into cytochrome c. Assay blanks were constructed as required for the experiment in question by using boiled enzyme, by omitting enzyme altogether, or by using bovine serum albumin instead of cytochrome c; identical low levels of methyl group incorporation were observed for each type of blank. After the incubation, the assay mixtures were cooled in an ice-water bath, and 85-1LI portions were pipetted onto disks (diameter, 25 mm) of filter paper (Schleicher and Schuell; no. 593-A) and were allowed to soak into the paper. Disks from a set of assays, which typically consisted of 20 to 25 samples, were then washed according to the following schedule: 200 ml of 10% trichloroacetic acid at 0°C for 10 min; 200 ml of 5% trichloroacetic acid at 95°C for 15 min; 150 ml of 5% trichloroacetic acid at room temperature for 5 min twice; 200 ml of ethanol at 70°C for 10 min; 150 ml of a mixture of ethanol and ether (1:1) at room temperature for 5 min; and 80 ml of ether at room temperature for 5 min. The disks were air dried for 10 min, placed in Nalgene scintillation tubes with 1 ml of scintillation fluid containing 4 g of 2,5-diphenyloxazole and 0.05 g of p-bis-[2-(4-methyl-S-phenyloxazolyl)]benzene per liter of toluene, sealed, and counted in a Packard Tri-Carb liquid scintillation counter. The counting efficiency of S-adenosyl-L-[methyl-'4C]methionine in the presence of assay ingredients under these conditions was determined to be 64% by comparison with the efficiency obtained in Aquasol scintillation fluid corrected for quench by using the standards supplied by Packard. Thus methyl-'4C-specific radioactivity was calculated to be 84 cpm/pmol. The specific activities of the enzyme preparations are expressed as picomoles of methyl group incorporated per 15 min per milligram of protein. Other methods. Protein determination was perforned by the dye-binding method of Bradford (1). The levels of cytochrome c were either estimated by low-temperature (-190°C) spectroscopic examination of intact cells (25a) or determined quantitatively by
855
spectrophotometric measurements of partially purified samples (29). Iso-l-cytochrome c from the strain D311-3A was isolated by the method used routinely in this laboratory (28). Oxygen uptake measurements were conducted with a Yellow Springs Instruments oxygen monitor (24) without subtraction of cyanideinsensitive consumption. Kinetic parameters were derived by the Lineweaver-Burk method (12). RESULTS Assay for cytochrome c methylase. A cytochrome c methylase activity was found to be present in crude extracts from several strains of bakers' yeast, S. cerevisiae. Incorporation of methyl groups from S-adenosyl-L-[methyl-14C]methionine into material insoluble in hot trichloroacetic acid and hot ethanol was dependent upon extract protein and cytochrome c substrate (Fig. 1), and the activity was abolished by boiling the extract for 5 min (data not shown). Methyl group incorporation in this reaction was time dependent (Fig. 2) and again required horse heart cytochrome c. The pH optimum of the 0.1 M Tris-hydrochloride buffer used in the incubation was 8.9, measured at room temperature; the actual pH in the reaction mixture was slightly lower since the S-adenosyl-L-[methyl"4C]methionine was used directly as supplied by the manufacturer in dilute H2SO4. The reaction
0.2 0.4 Extract Protein (mg)
FIG. 1. Cytochrome c-specific methylase activity and its dependence on extract protein. Crude extract supernatant was obtained from the normal strain D311-3A which was grown under derepressed conditions. The cells were disrupted with a Braun homogenizer, and methylase activity was determined with various amounts of extract protein and with 0.28 mg of either horse heart cytochrome c or bovine serum albumin (BSA) in 0.1-ml incubation mixtures containing S-adenosyl-L-[methyl-'4C]methionine. After 15 min of incubation, the amount of incorporation of "C-labeled methyl groups into material insoluble in hot trichloroacetic acid and hot ethanol was determined.
856
LIAO AND SHERMAN
J. BACTERIOL.
80 c
60
0 *
o0'a
zc.C *
20
0
30
60
90
Incubation Time (min) FIG. 2. Time course of the cytochrome c methylation reaction. Methylation activity was determined in a 0.5-mi reaction mixture containing 2.12 mg of crude extract proteins from the normal yeast D311-3A and the other components described in the legend to Fig. 1. At the times indicated, 0.06-ml portions were removed, and "C-labeled methyl group incorporation in the presence of horse heart cytochrome c or bovine serum albumin (BSA) was determined.
was not affected by the presence of up to 0.2 M NaCl, up to 50 mM MgCl2, up to 25 mM EDTA (disodium salt), or up to 14 mM 2-mercaptoethanol. The activity in strain D311-3A crude extract was stable for at least 1 week when stored at 40C and for at least 3 months when quickfrozen in liquid N2 and stored at -20°C, even if
the observation that cyanogen bromide cleavage peptides of horse heart cytochrome c, some not containing lysine 72, served as substrates for the purified N. crassa methylase (8). Native horse heart cytochrome c, which was devoid of trimethyllysine, was methylated by the yeast methylase, exhibiting a Km of 0.13 mM. We thus conclude that the methylation reaction is highly specific for cytochrome c. Coordinate regulation of cytochrome c and cytochrome c methylase. Due to catabolite repression, the level of cytochrome c, as well as those of all other mitochondrial cytochromes, are low in cells grown in glucose medium. Substantially higher levels are observed after depletion of glucose in the medium or by growth in media containing nonfermentable carbon sources (10). Similarly, anaerobically grown cells are deficient in their cytochromes and in their respiratory capacity (9). The diminished levels of cytochrome c due to catabolite repression and to anaerobiosis are correlated with lower specific activities of the cytochrome c-specific methylase (Table 1), suggesting a coordinate regulation of the synthesis Horse hrtT
q
40
cytochrofl.j
Lu Ot
*~30
20thawed and refrozen repeatedly. The apparent Km for S-adenosyl-L-methionine was 25 ,M; thus, the characteristics of yeast methylase are very similar to those described for 10the Neurospora enzyme (8, 15), except that the specific activity in crude extracts of D311-3A Yeat O under our conditions was only 15 to 30% of the sol-cytochrome E . E 0 activity reported for N. crassa. nv nl OA Aln Vt U.J U4 uz U.^S U.1 Specificity of the methylation reaction. Cytochrome c (mM) The specificity of the methylation reaction was FIG. 3. Specificity of the methylase reaction. Intested by using iso-l-cytochrome c from the creasing amounts of either horse heart or yeast (D311normal yeast strain D311-3A as a substrate, 3A) iso-1-cytochrome c were assayed for methyl-acsince this protein should be fully methylated cepting activity under the standard assay conditions and thus unable to accept more methyl groups with extracts from yeast strain D311-3A. The amount
in vitro. Amino acid analysis indeed confirmed the presence of one trimethyllysine residue per mol of iso-1-cytochrome c. As expected, this cytochrome c was a poor substrate for methylation in the standard assay (Fig. 3). The residual methyl group acceptance may be due to a small proportion of the preparation being incompletely methylated when isolated or to the methylation of denatured molecules at lysine residues other than lysine 72, a possibility suggested by
of incorporation of 4C-labeled methyl groups into hot trichloroacetic acid- and hot ethanol-insoluble material in the absence of added protein substrate (i.e., due to methylation of substrates present in the crude enzyme preparation) was less than 2pmol/15 min per mg and is subtracted from the results presented in this figure. Increasing amounts of bovine serum albumin did not stimulate methylation activity above this background. Results for horse heart cytochrome c are presented with the standard deviation of triplicate determinations.
CYTOCHROME c-SPECIFIC METHYLTRANSFERASE
VOL. 138, 1979
TABLE 1. Relative cytochrome c methylase activity, cytochrome c content, and respiratory capacity (oxygen uptake capacity) of the normal strain D3113A grown under various conditions upCondtionsOxygen capacConditiosm take ity(ld/h
Carbo
Carbon
Cyt-
chrome cc content
sp Methyl1ase 'act(methyf [C]methyl
15 min [dry (mg/g [dry per per mg of wt]) wt]) ~~protein) 100 (100)" 0.555 (100) 24.5 (100)
per mg
Ethanol Aerobic 36 (33) 0.275 (50) Glucose Aerobic 7.8 (7) 0.022 (4) Glucose Anaerobic a Numbers in parentheses are percentages.
12.1 (49) 5.2 (21)
of the substrate protein and the modifying activity. The extracts were mixed, and the resultant mixture was assayed to determine whether the reduction in methylase was indeed due to the lack of the enzyme and not to the presence of an inhibiting activity in the cell extract. As expected, intermediate activities were obtained, the total activity measured being the proportional sum of the activities in the individual extracts. It was also observed that, along with cytochrome c levels, the specific activity of methylase was somewhat higher in stationaryphase glucose-grown cells than in log-phase cells, again indicating parallel control. Further demonstration of the coordinate regulation of cytochrome c and methylase was obtained by following the adaptation of anaerobically grown cells to air and observing that the kinetics of induction of these activities were approximately parallel to each other and to the increasing capacity of the yeast to respire (Fig. 4). These results would be expected if the methylase were specific for cytochrome c and not generally required for the modification of other proteins. Normal cytochrome c methylase activity in cytochrome c-deficient mutants. Methylase levels were measured in extracts from a variety of normal and mutant strains of yeast. The mutant strains that were examined included p mutants derived from the two normal strains D273-1OB and D311-3A and cytochrome c-deficient mutants derived from these two normal strains or from the p- derivative of D273-1OB. In addition to the two isogenic series of mutants from D273-1OB and D311-3A, methylase activities were also examined in extracts from a third isogenic series of mutants derived from the strain p- cycl-1 CYC7-1, which contains a high level of iso-2-cytochrome c but completely lacks iso-1-cytochrome c. All of the strains were grown on the surface of nutrient medium under a condition that minimizes catabolite repression. Also,
857
all sets of determinations included an extract from D311-3A, and all determinations were related to the activity in this extract. There appeared to be variation in the amount of cytochrome c methylase activity among different normal strains. Some apparently normal strains contained less than 25% of the activity found in the normal strain D311-3A. However, the results presented in Table 2 clearly indicate that none of the cycl through cyc6 mutants are deficient in methylase activity, even though some of the mutants are almost devoid of cytochrome c. Although there was no difference in methylase activity between the p+ strain D311-3A and its p derivative D311-3A-1, the p+ strain D273-1OB contained approximately twice the activity of the p- derivative D273-1OB-1. It is possible that the deficiency observed in the p- derivative from D273-1OB but not in the p- derivative from D311-3A is similar to deficiencies of other components related to mitochondria that have been observed to be abnormally low in certain but not all p- strains (26). Without precise measurements of the relative proportion of methylated and unmethylated forms of cytochrome c, it is difficult to evaluate the full consequence of the variation of methylase activities among apparently normal strains and among certain p+ and p strains. 50 0
4-
Methylase
30 /-/
20!I
,
O
/
/
Cytochromec
e10 0~ 4 6 2 3 5 Time Aerated (hr) FIG. 4. Coordinate induction of cytochrome c and cytochrome c-specific methylase during aerobic adaptation. An anaerobically grown culture of strain D311-3A was subjected to respiratory adaptation, and portions were removed at various times for measurements of oxygen uptake capacity (QoJ,) cytochrome c content, and specific methylase activity. The results of all three parameters are expressed as the percentages of the levels found in log-phase cells grown aerobically in ethanol medium (see Table 1).
1
858
LIAO AND SHERMAN
J. BACTERIOL.
TABLE 2. Relative cytochrome c methylase activities and cytochrome c content in various isogenic series of normal and mutant strains of yeast Strain no.
Parent strain
Pertinent genotype
Relative Cytochome c Cyochrome C accontent ()' methylase tiVity (%)b
D311-3A Normal D311-3A-1 D311-3A pB-466 D311-3A cycl-17 B-467 D311-3A cycl-18 B-620 D311-3A cyc2-8 B-614 D311-3A cyc3-10 B-615 D311-3A cyc3-11 B-618 D311-3A cyc3-14 D273-1OB Normal D273-1OB-1 D273-1OB pB-271 D273-1OB cyc4-1 B-277 D273-1OB cyc5-1 B-320 D273-1OB cyc6-1 B-326 D273-1OB-1 p- cyc2-5 B-361 D273-1OB-1 p- cyc4-5 B-4927 p- cycl-1 CYC7-1 B-4736 B-4927 p- cyc2-1O cycl-1 CYC7-1 B-4764 B-4927 p- cyc3-17 cycl-1 CYC7-1 B-4766 B-4927 p- cyc3-19 cycl-l CYC7-1 a The cytochrome c levels were estimated semi-quantitatively by low-temperature examination of intact cells. b Compared with the specific activity of D311-3A extracts.
DISCUSSION
We have demonstrated that the yeast S. cerevisiae contains a cytochrome c-specific methylase with properties similar to those of the analogous enzyme purified from another fungus, N. crassa (8, 15). We also showed that this activity is reduced in cells grown under catabolite repression or grown anaerobically when the level of cytochrome c is also reduced and that it is induced during anaerobic-to-aerobic adaptation approximately in parallel with the induction of cytochrome c. These results suggest that the methylase is related to the formation of cytochrome c and that it is not required for other proteins, or at least proteins not under the same general control as cytochrome c. The normal level of cytochrome c-methylase in mutants completely or almost completely deficient in cytochrome c establishes that the cytochrome c level itself does not directly determine the methylase activity. Thus, it appears that the methylase and cytochrome c as well as other mitochondrial cytochromes and respiratory components are under the same regulatory control. All of the cytochrome c-deficient cyc mutants screened in this investigation, encompassing strains carrying mutations at all of the known loci that affect cytochrome c levels or function, were found to contain normal levels of methylase activity, thus suggesting that mutants deficient in methylase may not necessarily also be defi-
100 100 100 97 5 104 5 116 20 85 10 129 5 99 5 100 100 120 100 56 50 73 80 82 40 91 40 79 50 50 100 27
JouRNAL oF BACrzIRoLOGY, June 1979, p. 863-0 0021-9193/79/060863/08$02.00/0
Yeast Cytochrome c-Specific Protem-Lysine Methyltransferase: Coordinate Regulation with Cytochrome c and Activities in cyc Mutantst HANS H. LIAOt AND FRED SHERMAN* Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
Received for publication 17 January 1979
The cytochromes c of fungi and higher plants contain one or two residues of e-N-trimethyllysine, whose biological role is unknown. A cytochrome c-specific Sadenosylmethionine:protein-lysine methyltransferase (methylase) activity was shown to be present in extracts of the bakers' yeast Saccharomyces cerevisiae, and basic kinetic properties of this enzyme are described. The specific activity of the methylase was lower in extracts of cells grown under conditions of catabolite (glucose) repression or anaerobiosis where cytochrome c levels were low, compared with cells grown under derepressed conditions where cytochrome c levels were high. During anaerobic-to-aerobic adaptation, the methylase was induced in parallel with cytochrome c, thus suggestiDng that the syntheses of cytochrome c and cytochrome c methylase are coordinately regulated. None of the cyc strains surveyed (cycl, cyc2, cyc3, cyc4, cyc5, and cyc6) had diminished levels of methylase, although some of them were completely or almost completely deficient in cytochrome c.
Cytochrome c isolated from fungi and higher plants has been found to contain e-N-trimethyllysine (4). Cytochrome c from Neurospora crassa (4), both iso-cytochromes c from the bakers' yeast Saccharomyces cerevisiae (5), as well as cytochromes c from other fungi (2, 6, 16) have e-N-trimethyllysine confined to a single position corresponding to the position of amino acid 72 in vertebrate cytochrome c, whereas cytochromes c from wheat germ (4) and other higher plants (2, 6, 16) have two e-N-trimethyllysine residues confined to positions 72 and 86. In contrast, vertebrate cytochromes c do not contain any trimethyllysine residues. Protein methylation, which is widespread in nature but of unknown functional significance (2, 16), is a posttranslational event, with the methyl group derived from S-adenosylmethionine. In N. crassa, a precursor-product relationship has been established between the unmethylated and methylated cytochromes c (22); chromatographic separation of the cytochromes c from S. cerevisiae also revealed, in addition to the major iso-l-cytochrome c and minor iso-2cytochrome c methylated proteins, a small
amount of the unmethylated forms (11), which are presumably the precursors of the methylated cytochromes c. Furthermore, a cytochrome c-
specific S-adenosylmethionine:protein-lysine methyltransferase has been purified recently from N. crassa (8) and shown to restrict methylation to the lysine residue at position 72 of native horse heart cytochrome c, thus appearing to be the enzyme responsible for the modification of cytochrome c in vivo. The biological function of cytochrome c methylation is unknown. Measurement of the stabilities toward denaturants (17), circular dichroic spectra (13), and kinetic parameters in reactions with cytochrome c peroxidase, oxidase, and succinate oxidoreductase (18) showed only minor, if any, differences between the methylated and unmethylated yeast iso-1-cytochromes c. However, recent evidence suggests that trimethylated iso-1-cytochrome c does have a greater affinity for mitochondria than does the unmethylated form (19); although the in vivo significance of this finding is unclear, it suggested that
t Report UR-3490-1496 from the U. S. Department of Energy at the University of Rochester, Department of Radiation Biology and Biophysics. t Present address: Department of Bacteriology and Immunology, University of California, Berkeley, CA 94720.
853
the lack of such modification may express itself phenotypically as impaired cytochrome c function. Therefore, in the investigation reported here, cyc mutants of yeast, which are deficient in cytochrome c levels and activities (7, 23, 27, 28), were screened for cytochrome c methylase activity, with the goal of possibly identifying a
854
LIAO AND SHERMAN
methylase-deficient strain among the cyc mutants and demonstrating a role for the modification in vivo. (The term methylase refers to the enzyme activity that transfers methyl groups from S-adenosylmethionine to proteins.) In addition, we investigated whether the enzyme was subject to the same regulatory constraints as cytochrome c, i.e. repression of synthesis by glucose (10) and anaerobiosis (9) and derepression by nonfermentable carbon sources and aerobiosis. If the methylase were specific for cytochrome c and necessary for its function, it was expected that both of these proteins would be coordinately regulated. MATERIAIS AND METHODS Materials. S-Adenosyl-L-[methyl- 14C]methionine (58 to 59 mCi/mmol) was obtained from Amersham/ Searle, horse heart cytochrome c was obtained from Sigma Chemical Co., and Aquasol scintillation fluid was obtained from New England Nuclear Corp. Strains of S. cerevisiae. The strains examined in this study included the normal strains D311-3A and D273-1OB and cytochrome c-deficient mutants derived from these normal strains by using a spectroscopic scanning procedure (23), a benzidine staining procedure (28) and a chlorolactate enrichment procedure (27). Also examined were the cycl-1 CYC7-1 strain B4927, which functions normally, although it contains a high level of exclusively iso-2-cytochrome c, and cytochrome c-deficient mutants derived from this strain (7). Mutation at the structural gene cycl results in deficiencies in the amount or function of iso-icytochrome c; mutations at any of the loci from cyc2 through cyc6 result in various degrees of deficiency of both iso-l-cytochrome c and iso-2-cytochrome c. Growth of yeast. Small quantities of yeast sufficient for the measurement of methylase activity were obtained by the growth of each strain on the surface of a single plate containing 1% yeast extract (Difco), 2% peptone (Difco), 2% agar, and either 1% sucrose or 2% glucose. Yeast were streaked as thin lines across the surface of the plates, a growth condition that allows for derepression of the cytochromes (27). After 3 days of incubation at 30°C, the cells were scraped off the plate, washed twice with distilled water, and stored at -30°C. Larger amounts of cells were obtained by growth in 10-liter batches of medium containing 1% Ardamine Z yeast extract, 2% technicalgrade peptone (Difco), 0.2% (vol/vol) polypropylene glycol, 34 mg of penicillin per liter, 100 mg of streptomycin per liter, and either 2% (vol/vol) ethanol or 5% glucose. Cultures were grown in 14-liter Fermentation Design fermentors at 30°C with stirring at 500 rpm and with aeration of 600 liters of air per min. The cells were harvested at 4°C by centrifugation in a refrigerated Lourdes 30-R Clini-Fuge centrifuge for 6 min at 2,000 rpm, washed twice with water, and stored at -300C. Anarobic cultures were obtained by growth at 300C in 12 liters of a medium containing 1% yeast extract (Difco), 3% glucose, 0.1% KH2PO4, 0.12% (NH4)2SO4, 42 ml of a solution of 0.29% ergosterol and 28% (vol/
J. BACTrERIOL.
vol) Tween 80 in absolute ethanol (resulting in final concentrations of 10 mg/liter and 1 ml/liter, respectively) (3), 100 mg of streptomycin per liter, 34 mg of penicillin per liter, and 0.25% (vol/vol) polypropylene glycol. Each of the cultures was inoculated with a 500ml preculture grown with minimum aeration in a volumetric flask filled to the neck. The 12-liter cultures were maintained air-free by continuous flushing with N2 (Matheson prepurified N2, further scrubbed of 02 by a model 4002 Oxy-Trap [Alltech Associates]) at 80 ml/min for 6 h before inoculation and throughout growth; the cultures were agitated by stirring at 500 rpm. After 18 h, the cultures were chilled to 40C, transferred to chilled centrifuge bottles by displacement with N2, and centrifuged as described above. Cells were washed twice with cold deaerated water and either used immediately for the adaptation experiment described below or stored at -30°C for the subsequent preparation of crude extract. Respiratory adaptation. Adaptation of anaerobically grown yeast to air was performed by suspending cells to a concentration of 16 g (wet weight) per liter in 8 liters of 40 mM potassium phosphate (pH 7.4), 0.3% glucose, 1% (vol/vol) ethanol, and 0.2% (vol/ vol) polypropylene glycol. The culture was aerated at 28°C in a 14-liter Fermentation Design unit with 600 liters of air per min while being stirred at 500 rpm (20). Portious of the culture were obtained by siphoning 1.5 liters into centrifuge bottles containing ice and centrifuging in a cold Lourdes Clini-Fuge centrifuge as described above. The pelleted yeast cells were washed with cold water and recentrifuged in a Beckman JS-13 rotor for 5 min at 6,000 rpm; they were then either resuspended in water for the determination of oxygen uptake capacity and cytochrome c content or stored at -30°C for the eventual measurement of methylase activity. Preparation of crude extracts. Crude extracts of yeast were prepared by disruption with glass beads in a Braun type 2876 homogenizer. Ten-gram (wet weight) batches were suspended with 20 ml of 0.1 M Tris-hydrochloride buffer (pH 8.9) and shaken for a total of 90 s with 20 g of beads (diameter, 0.45 to 0.5 mm) in a Teflon bottle kept cool by a jet of CO2 gas. The extract was decanted from the beads and freed of cell debris by centrifuging twice at 4°C for 15 min in a Beckman JA-20 rotor spun at 15,000 rpm in a Beckman J-21B centrifuge. Efficiency of cell breakage was estimated by inspection under the microscope to be greater than 95%. For the preparation of extracts from small quantities of cells grown on solid media, an adapter for the Braun homogenizer was custom designed and constWcted in our facilities. The adapter accommodates up to eight Eppendorf 1.5-ml polypropylene micro test tubes with attached caps in two tiers and was constructed of aluminum to allow efficient cooling by CO2 gas. Screws holding the two tiers of racks together also served to prevent the adapter itself from rotating during shaking of the yeast suspension. Optimal breakage was achieved by suspending 0.4 g (wet weight) of yeast with 1 g of glass beads in sufficient 0.1 M Trishydrochloride buffer (pH 8.9) to fill the micro test tube and shaking the cell suspension for a total of 2.5 min. The methylase-containing supernatant from the
VOL. 138, 1979
CYTOCHROME c-SPECIFIC METHYLTRANSFERASE
crude extract was obtained after centrifuging twice at 40C in an Eppendorf 3200 centrifuge, the first spin being performed in the same test tube used for cell breakage. The efficiency of breakage in the micro test tubes was only about 50%; variations in the proportion of yeast to glass beads did not improve the extraction since a lower ratio of yeast to glass beads (0.2:1) yielded an extract too dilute for the methylase assay, whereas higher ratios (1:1 or 1.4:1) diminished the breakage efficiency still further. Assay for cytochrome c-specific methylase. The assay procedure was adapted from that described for the enzyme in extracts of N. crassa (15) but was simplified by using paper filter disks for the collection of trichloroacetic acid-precipitated protein (14). A final volume of 0.1 ml of each reaction mixture consisted of 0.1 M Tris-hydrochloride (pH 8.9), 0.225 ,Ci of Sadenosyl-L-[methyl-'4C]methionine (used without dilution of the commercial product), 0.23 mM horse heart cytochrome c, unless otherwise stated, and various amounts of crude extracts. The reactions were carried out at 37°C, usually for 15 min. Preincubation of the mixture for 5 min before the addition of Sadenosyl-L-[methyl-'4C]methionine had no effect on the subsequent incorporation of methyl-'4C into cytochrome c. Assay blanks were constructed as required for the experiment in question by using boiled enzyme, by omitting enzyme altogether, or by using bovine serum albumin instead of cytochrome c; identical low levels of methyl group incorporation were observed for each type of blank. After the incubation, the assay mixtures were cooled in an ice-water bath, and 85-1LI portions were pipetted onto disks (diameter, 25 mm) of filter paper (Schleicher and Schuell; no. 593-A) and were allowed to soak into the paper. Disks from a set of assays, which typically consisted of 20 to 25 samples, were then washed according to the following schedule: 200 ml of 10% trichloroacetic acid at 0°C for 10 min; 200 ml of 5% trichloroacetic acid at 95°C for 15 min; 150 ml of 5% trichloroacetic acid at room temperature for 5 min twice; 200 ml of ethanol at 70°C for 10 min; 150 ml of a mixture of ethanol and ether (1:1) at room temperature for 5 min; and 80 ml of ether at room temperature for 5 min. The disks were air dried for 10 min, placed in Nalgene scintillation tubes with 1 ml of scintillation fluid containing 4 g of 2,5-diphenyloxazole and 0.05 g of p-bis-[2-(4-methyl-S-phenyloxazolyl)]benzene per liter of toluene, sealed, and counted in a Packard Tri-Carb liquid scintillation counter. The counting efficiency of S-adenosyl-L-[methyl-'4C]methionine in the presence of assay ingredients under these conditions was determined to be 64% by comparison with the efficiency obtained in Aquasol scintillation fluid corrected for quench by using the standards supplied by Packard. Thus methyl-'4C-specific radioactivity was calculated to be 84 cpm/pmol. The specific activities of the enzyme preparations are expressed as picomoles of methyl group incorporated per 15 min per milligram of protein. Other methods. Protein determination was perforned by the dye-binding method of Bradford (1). The levels of cytochrome c were either estimated by low-temperature (-190°C) spectroscopic examination of intact cells (25a) or determined quantitatively by
855
spectrophotometric measurements of partially purified samples (29). Iso-l-cytochrome c from the strain D311-3A was isolated by the method used routinely in this laboratory (28). Oxygen uptake measurements were conducted with a Yellow Springs Instruments oxygen monitor (24) without subtraction of cyanideinsensitive consumption. Kinetic parameters were derived by the Lineweaver-Burk method (12). RESULTS Assay for cytochrome c methylase. A cytochrome c methylase activity was found to be present in crude extracts from several strains of bakers' yeast, S. cerevisiae. Incorporation of methyl groups from S-adenosyl-L-[methyl-14C]methionine into material insoluble in hot trichloroacetic acid and hot ethanol was dependent upon extract protein and cytochrome c substrate (Fig. 1), and the activity was abolished by boiling the extract for 5 min (data not shown). Methyl group incorporation in this reaction was time dependent (Fig. 2) and again required horse heart cytochrome c. The pH optimum of the 0.1 M Tris-hydrochloride buffer used in the incubation was 8.9, measured at room temperature; the actual pH in the reaction mixture was slightly lower since the S-adenosyl-L-[methyl"4C]methionine was used directly as supplied by the manufacturer in dilute H2SO4. The reaction
0.2 0.4 Extract Protein (mg)
FIG. 1. Cytochrome c-specific methylase activity and its dependence on extract protein. Crude extract supernatant was obtained from the normal strain D311-3A which was grown under derepressed conditions. The cells were disrupted with a Braun homogenizer, and methylase activity was determined with various amounts of extract protein and with 0.28 mg of either horse heart cytochrome c or bovine serum albumin (BSA) in 0.1-ml incubation mixtures containing S-adenosyl-L-[methyl-'4C]methionine. After 15 min of incubation, the amount of incorporation of "C-labeled methyl groups into material insoluble in hot trichloroacetic acid and hot ethanol was determined.
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LIAO AND SHERMAN
J. BACTERIOL.
80 c
60
0 *
o0'a
zc.C *
20
0
30
60
90
Incubation Time (min) FIG. 2. Time course of the cytochrome c methylation reaction. Methylation activity was determined in a 0.5-mi reaction mixture containing 2.12 mg of crude extract proteins from the normal yeast D311-3A and the other components described in the legend to Fig. 1. At the times indicated, 0.06-ml portions were removed, and "C-labeled methyl group incorporation in the presence of horse heart cytochrome c or bovine serum albumin (BSA) was determined.
was not affected by the presence of up to 0.2 M NaCl, up to 50 mM MgCl2, up to 25 mM EDTA (disodium salt), or up to 14 mM 2-mercaptoethanol. The activity in strain D311-3A crude extract was stable for at least 1 week when stored at 40C and for at least 3 months when quickfrozen in liquid N2 and stored at -20°C, even if
the observation that cyanogen bromide cleavage peptides of horse heart cytochrome c, some not containing lysine 72, served as substrates for the purified N. crassa methylase (8). Native horse heart cytochrome c, which was devoid of trimethyllysine, was methylated by the yeast methylase, exhibiting a Km of 0.13 mM. We thus conclude that the methylation reaction is highly specific for cytochrome c. Coordinate regulation of cytochrome c and cytochrome c methylase. Due to catabolite repression, the level of cytochrome c, as well as those of all other mitochondrial cytochromes, are low in cells grown in glucose medium. Substantially higher levels are observed after depletion of glucose in the medium or by growth in media containing nonfermentable carbon sources (10). Similarly, anaerobically grown cells are deficient in their cytochromes and in their respiratory capacity (9). The diminished levels of cytochrome c due to catabolite repression and to anaerobiosis are correlated with lower specific activities of the cytochrome c-specific methylase (Table 1), suggesting a coordinate regulation of the synthesis Horse hrtT
q
40
cytochrofl.j
Lu Ot
*~30
20thawed and refrozen repeatedly. The apparent Km for S-adenosyl-L-methionine was 25 ,M; thus, the characteristics of yeast methylase are very similar to those described for 10the Neurospora enzyme (8, 15), except that the specific activity in crude extracts of D311-3A Yeat O under our conditions was only 15 to 30% of the sol-cytochrome E . E 0 activity reported for N. crassa. nv nl OA Aln Vt U.J U4 uz U.^S U.1 Specificity of the methylation reaction. Cytochrome c (mM) The specificity of the methylation reaction was FIG. 3. Specificity of the methylase reaction. Intested by using iso-l-cytochrome c from the creasing amounts of either horse heart or yeast (D311normal yeast strain D311-3A as a substrate, 3A) iso-1-cytochrome c were assayed for methyl-acsince this protein should be fully methylated cepting activity under the standard assay conditions and thus unable to accept more methyl groups with extracts from yeast strain D311-3A. The amount
in vitro. Amino acid analysis indeed confirmed the presence of one trimethyllysine residue per mol of iso-1-cytochrome c. As expected, this cytochrome c was a poor substrate for methylation in the standard assay (Fig. 3). The residual methyl group acceptance may be due to a small proportion of the preparation being incompletely methylated when isolated or to the methylation of denatured molecules at lysine residues other than lysine 72, a possibility suggested by
of incorporation of 4C-labeled methyl groups into hot trichloroacetic acid- and hot ethanol-insoluble material in the absence of added protein substrate (i.e., due to methylation of substrates present in the crude enzyme preparation) was less than 2pmol/15 min per mg and is subtracted from the results presented in this figure. Increasing amounts of bovine serum albumin did not stimulate methylation activity above this background. Results for horse heart cytochrome c are presented with the standard deviation of triplicate determinations.
CYTOCHROME c-SPECIFIC METHYLTRANSFERASE
VOL. 138, 1979
TABLE 1. Relative cytochrome c methylase activity, cytochrome c content, and respiratory capacity (oxygen uptake capacity) of the normal strain D3113A grown under various conditions upCondtionsOxygen capacConditiosm take ity(ld/h
Carbo
Carbon
Cyt-
chrome cc content
sp Methyl1ase 'act(methyf [C]methyl
15 min [dry (mg/g [dry per per mg of wt]) wt]) ~~protein) 100 (100)" 0.555 (100) 24.5 (100)
per mg
Ethanol Aerobic 36 (33) 0.275 (50) Glucose Aerobic 7.8 (7) 0.022 (4) Glucose Anaerobic a Numbers in parentheses are percentages.
12.1 (49) 5.2 (21)
of the substrate protein and the modifying activity. The extracts were mixed, and the resultant mixture was assayed to determine whether the reduction in methylase was indeed due to the lack of the enzyme and not to the presence of an inhibiting activity in the cell extract. As expected, intermediate activities were obtained, the total activity measured being the proportional sum of the activities in the individual extracts. It was also observed that, along with cytochrome c levels, the specific activity of methylase was somewhat higher in stationaryphase glucose-grown cells than in log-phase cells, again indicating parallel control. Further demonstration of the coordinate regulation of cytochrome c and methylase was obtained by following the adaptation of anaerobically grown cells to air and observing that the kinetics of induction of these activities were approximately parallel to each other and to the increasing capacity of the yeast to respire (Fig. 4). These results would be expected if the methylase were specific for cytochrome c and not generally required for the modification of other proteins. Normal cytochrome c methylase activity in cytochrome c-deficient mutants. Methylase levels were measured in extracts from a variety of normal and mutant strains of yeast. The mutant strains that were examined included p mutants derived from the two normal strains D273-1OB and D311-3A and cytochrome c-deficient mutants derived from these two normal strains or from the p- derivative of D273-1OB. In addition to the two isogenic series of mutants from D273-1OB and D311-3A, methylase activities were also examined in extracts from a third isogenic series of mutants derived from the strain p- cycl-1 CYC7-1, which contains a high level of iso-2-cytochrome c but completely lacks iso-1-cytochrome c. All of the strains were grown on the surface of nutrient medium under a condition that minimizes catabolite repression. Also,
857
all sets of determinations included an extract from D311-3A, and all determinations were related to the activity in this extract. There appeared to be variation in the amount of cytochrome c methylase activity among different normal strains. Some apparently normal strains contained less than 25% of the activity found in the normal strain D311-3A. However, the results presented in Table 2 clearly indicate that none of the cycl through cyc6 mutants are deficient in methylase activity, even though some of the mutants are almost devoid of cytochrome c. Although there was no difference in methylase activity between the p+ strain D311-3A and its p derivative D311-3A-1, the p+ strain D273-1OB contained approximately twice the activity of the p- derivative D273-1OB-1. It is possible that the deficiency observed in the p- derivative from D273-1OB but not in the p- derivative from D311-3A is similar to deficiencies of other components related to mitochondria that have been observed to be abnormally low in certain but not all p- strains (26). Without precise measurements of the relative proportion of methylated and unmethylated forms of cytochrome c, it is difficult to evaluate the full consequence of the variation of methylase activities among apparently normal strains and among certain p+ and p strains. 50 0
4-
Methylase
30 /-/
20!I
,
O
/
/
Cytochromec
e10 0~ 4 6 2 3 5 Time Aerated (hr) FIG. 4. Coordinate induction of cytochrome c and cytochrome c-specific methylase during aerobic adaptation. An anaerobically grown culture of strain D311-3A was subjected to respiratory adaptation, and portions were removed at various times for measurements of oxygen uptake capacity (QoJ,) cytochrome c content, and specific methylase activity. The results of all three parameters are expressed as the percentages of the levels found in log-phase cells grown aerobically in ethanol medium (see Table 1).
1
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LIAO AND SHERMAN
J. BACTERIOL.
TABLE 2. Relative cytochrome c methylase activities and cytochrome c content in various isogenic series of normal and mutant strains of yeast Strain no.
Parent strain
Pertinent genotype
Relative Cytochome c Cyochrome C accontent ()' methylase tiVity (%)b
D311-3A Normal D311-3A-1 D311-3A pB-466 D311-3A cycl-17 B-467 D311-3A cycl-18 B-620 D311-3A cyc2-8 B-614 D311-3A cyc3-10 B-615 D311-3A cyc3-11 B-618 D311-3A cyc3-14 D273-1OB Normal D273-1OB-1 D273-1OB pB-271 D273-1OB cyc4-1 B-277 D273-1OB cyc5-1 B-320 D273-1OB cyc6-1 B-326 D273-1OB-1 p- cyc2-5 B-361 D273-1OB-1 p- cyc4-5 B-4927 p- cycl-1 CYC7-1 B-4736 B-4927 p- cyc2-1O cycl-1 CYC7-1 B-4764 B-4927 p- cyc3-17 cycl-1 CYC7-1 B-4766 B-4927 p- cyc3-19 cycl-l CYC7-1 a The cytochrome c levels were estimated semi-quantitatively by low-temperature examination of intact cells. b Compared with the specific activity of D311-3A extracts.
DISCUSSION
We have demonstrated that the yeast S. cerevisiae contains a cytochrome c-specific methylase with properties similar to those of the analogous enzyme purified from another fungus, N. crassa (8, 15). We also showed that this activity is reduced in cells grown under catabolite repression or grown anaerobically when the level of cytochrome c is also reduced and that it is induced during anaerobic-to-aerobic adaptation approximately in parallel with the induction of cytochrome c. These results suggest that the methylase is related to the formation of cytochrome c and that it is not required for other proteins, or at least proteins not under the same general control as cytochrome c. The normal level of cytochrome c-methylase in mutants completely or almost completely deficient in cytochrome c establishes that the cytochrome c level itself does not directly determine the methylase activity. Thus, it appears that the methylase and cytochrome c as well as other mitochondrial cytochromes and respiratory components are under the same regulatory control. All of the cytochrome c-deficient cyc mutants screened in this investigation, encompassing strains carrying mutations at all of the known loci that affect cytochrome c levels or function, were found to contain normal levels of methylase activity, thus suggesting that mutants deficient in methylase may not necessarily also be defi-
100 100 100 97 5 104 5 116 20 85 10 129 5 99 5 100 100 120 100 56 50 73 80 82 40 91 40 79 50 50 100 27
