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Cytochrome C methylation
MINIREVIEW CYTOCHROME
C METHYLATION
ENRICO POLASTRO*, ARTHUR G. SCIHNECK*, JOSE LEONIS*. SANGDUK KIM and WOON
Kr PAIK
Chimie
Generale 1. Faculte des Sciences, Universite Libre de Bruxelles. Belgique*. and Fels Research Institute and Department of Biochemistry, Temple University School of Medicine. Philadelphia, PA. 19140, U.S.A. (Receioed 29 March 1978)
Abstract-Enzymatic methylation of 72 lysyl residue of cytochrome c in lower organisms facilitates its binding to mitochondria, and subsequently plays an important role in the electron transport process.
Protein methylation is an ubiquitously occurring post-translational modification reaction involving lysine, arginine, dicarboxylic amino acids and histidine side chains (Paik & Kim, 1975). Biochemical significance of the reaction is not generally well understood. It is however being actively investigated in the area of bacterial chemotaxis (Kleene et al., 1977; Werf & Koshland, 1977), processing of pituitary hormones (Diliberto & Axelrod, 1974), exocytosis (Diliberto et al.. 1976), gene regulation (Byvoet & Baxter, 1975; Paik & Kim, 1974), and muscle contraction (Morse et al.. 1975; Huszar, 1975). Recent evidence strongly indicates that carnitine, which is an acyl carrier within the mitochondrial membrane, is synthesized from l-N-trimethyllysine and that the formation of E-N-trimethyllysine results from enzymatic methylation of protein-lysine residues using S-adenosyl-L-methionine and subsequent degradation by proteolytic enzymes (LaBadie et al., 1976; Paik & Kim, 1977). In this review, we present evidence which indicates that enzymatic trimethylation of cytochrome c of yeast or Neurospora crassa facilitates the binding of the cytochrome c with mitochondria, and subsequently play an important role in the process of electron transport. HISTORICAL BACKGROUND Cytochrome
c is a universally
occurring
protein,
found
in the mitochondrion of all eukaryotes. Because of its universality and ease of preparation, many studies have been devoted to its primary and tertiary structure. In fact. cytochrome c is one of the proteins most thoroughly studied. and cytochrome c from up to 100 species of microorganisms, plants, and animals have been completely sequenced (Dayhoff et al., 1972). Since the biological role of cytochrome c remained the same throughout evolution. one would expect that the primary structure of the protein from various sources should not differ too far from each other. However, it was found that the cytochrome c in man has diverged from the cytochrome c in Neurospora crassa in 44 out of 104 places (DeLange et al., 1969), yet approximately 34 amino acid residues remaining invariant. Among the invariant amino acid residues, 795
the undecapeptide of residues 70 and 80 is the largest sequence in all the cytochrome c (Margoliash & Schejter, 1966). Thus, this peptide seems to be of prime importance for the function of the protein, any mutation in it being probably lethal. In 1969, DeLange et al. demonstrated that the 70-80 undecapeptide of the cytochrome c is not absolutely invariant in all species: Cytochrome c of wheat germ and Neurospora crussa were found to contain c-N-trimethyllysine residue in place of lysine at Res-72 position (1969). Since then, the cytochrome ( of many other organisms also were found to contain this residue; for example, Neurosporu c~u.w~. Succharomyces crrecisiae. and Cundida krusei contain a single C-N-trimethyllysine at Res-72, while wheat germ, Spinacea oleracea (spinach) and AIltmm porrutn (leak) contain 2 e-N-trimethyllysine at Res-72 and Res-86 (Brown & Boulter, 1973; Brown et al.. 1973; DeLange et al., 1970; Ramshaw & Boulter, 1975). However, the following organisms contain no methylated lysine: Lamprey, dogfish, bullfrog, turtle, rattlesnake, turkey. mammals and insect (DeLange et al.. 1970). These findings indicate a sharp evolutionary differentiation in the development of specific methylation enzymes among Ascotnycetes, higher plants, vertebrates and insects. In N. crassa, there are two species of cytochrome c, namely C, and C,,, differing only at the stage of residues 72, this position being occupied by a E-N-trimethyllysine in C, and a lysine in C,, (Scott & Mitchell. 1969). Working with N. crassa poky mutant (mi-l), Scott and Mitchell demonstrated by the use of pulse-chase experiment that cytochrome C,, form was first formed and then converted to C, on ageing (Scott & Mitchell, 1969). This observation strongly suggested that methylation of the c-amino group of lysine residue occurs on the whole cytochrome c polypeptide level. In contrast to the case of N. crassa. in Saccharomyces cereuisiae. where the unmethylated form of cytochrome c coexists in vic’o with the methylated form (Foucher et al., 1972). Brunko could not find any evidence of in bico conversion of the unmethylated cytochrome c to the methylated (Brunko. 1972). He has consequently suggested that cyto-
ENKIC~ POLASTKO cf ul.
796 Table
I. Substrate
Source of cytochromc Horse Rabbit Beef Guanaco Mouse Pigeon Chicken Tuna
activity
of various
cytochromes
c for protein-lysine
Moles of t-N-trimethyllysinc mole of cytochrome c irr ~~ilo
(’
(Llama)
to 1.26 nmole
of [methyl-‘4C]
I oo*
I I? II? X6 57
I20 66 96 40 166 -I incorporated/min/mg
ENZYMOLOGY
In order to investigate this specific methylation of lysine Res-72 in the cytochrome c, a protein methylase III [S-Adenosylmethionine:protein-lysine methyltransferase; EC 2. I. I.431 was recently purified from N. crassa approximately 3500-fold (Nochumson YI al.. 1977: Durban et al.. 1978). The enzyme is highly specific in its protein substrate requirement; of all proteins tested. only cytochrome c had substrate activity. Cytochrome c from various species were able to act as methyl accepting protein substrates in a range of 5@150”, compared to horse heart cytochrome c (Table I). Only one cytochrome c species was a very poor substrate, iso-l of baker’s yeast. This finding supports the notion that only unmethylated cytochrome c can act as methyl acceptor and once position 72 is methylated. the protein-lysine methyltransferase can not add any further methyl groups, The purified protein methylase III of N. Crassa appears to recognize the amino acid sequence of -XLys-Lys-Y- where X and Y may vary but a double lysine sequence is of absolute necessity (Durban er al., 1978). Even though I9 lysine residues are present in 104 amino acid residues of horse heart cytochrome
* N.A. represents
tion
site at lysine
not active
protein.
residues
7 8 not
of horse heart cytochrome (Durban ct al.. 1978)
K, (mM)
Cytochrome c (horse heart): Native Ethanol-denatured Heme-free CNBr-peptide: Peptide I (residues l-80) Peptide II (residues l-65) Peptide III (residues 666104) Peptide IV (residues 81-104) Peptide V (residues 66-80)
of enzyme
c, only single lysine residue at position 72 is methylated, which coincides with the lysine found methylated irl r:ica in cytochrome c of N. crassa. Horse heart cytochrome c contains 2 methionyl residues at position 65 and 80; however, treatment of the protein with cyanogen bromide in 0.1 N HCI yielded five peptides due to incomplete reaction. consisting of residues I-80. residues l-65. residues 66-104. residues 8l-- 104, and residues 6680 (peptides I, II. III. IV and V, respectively (Durban et al., 1978). Peptide I. II and III were good substrate for the enzyme. having low K, values. while peptide IV and V were completely inactive as methyl-accepting substrate (Table 2). The results strongly indicate that not only the reactive site such as -X-Lys-Lys-Y- sequence is necessary, but also the length of the polypeptide chain is an important feature in determining methylaccepting activity. When present in the intact molecule of horse heart cytochrome c, peptide 11 (residues l-65) cannot be methylated with S-adenosyl-L-methionine by the purified protein-lysine methyltransferase (protein methylase III) (Durban et ul.. 1978). However. the peptide served as an excellent substrate for the enzyme (with high V,,, and low K, value; Table 2) and further studies indicated that chymotryptic peptide containing residue 7 lysine (-X-Lys.‘7-Lys/8-Y-) was the only one methylated. The reason for the potential methyla-
Parameters of various CNBr-peptides protein-lysine methyltransferase
tested
methvl accepting activ;ty
0
chrome c methylation in S. crrrcisiuc~ occurs on the nascent polypeptide stage, analogous to the enzymatic methylation of myosin (Morse er a/.. 1975).
2. Kinetic
of iz’. c’r’uss~r
0 0 0 0 0 0 0 0
* Corresponds
Substrate
Relative
0
Frog Silkworm Baker’s yeast
Table
methyltransferasc
0.32 _+ 0.13 0.20 0.03 0.007 0.04 0.04 N.A.* N.A.
being
methylated
c for !V. cr~z.w
V ,“.I\ (nmole methyl~min;mg emyme protein)
‘4 _+ h 29 7 I6 27 2.4 N.A. N.A.
Cytochrome c methylation in intact cytochrome c, particularly “native” one, may be due to the fact that if the enzyme binds its protein substrate at the “front” orientation of the molecule, the lysine residues 778 are positioned in back essentially hidden from the enzyme (Durban rt al.. 1978). Thus, in peptide fragment II, the constraints imposed on lysine residue 7-8 as part of native cytochrome c are removed and this methylation site now becomes freely accessible to the enzyme for methylation. Contrary to the irr oivo product, the methylation products of horse heart cytochrome c by the purified N. crassa protein-lysine methyltransferase showed three methylated amino acids, i.e. E-N-monomethyllysine, l-N-dimethyllysine and l-N-trimethyllysine in a ratio of 1:3:4 (Durban er al., 1978). This ratio remained constant throughout 3500-fold purification, suggesting that a single enzyme is responsible for methylation of all three lysines by stepwise mechanism. The optimum pH of the enzyme is 9.0, its pl is 4.8, and its molecular weight is about 120,000. The K, value for cytochrome c is 0.3 mM. the K, for S-adenosyl-t.-methionine is 19 PM, while the inhibition constant K, for S-adenosyl-t_-homocysteine is about 2pM. It is also of interest to note that histone did not accept methyl groups from S-adenosyl-L-methionine with the N. crmsu enzyme (Durban rt a/.. 1978). This is in sharp contrast to the enzyme from calf thymus or rat brain which preferentially acts on histones (Paik & Kim. 1970; Duerre et al.. 1977). while cytochrome c is a poor substrate. Since wheat germ cytochrome c contains two l-N-trimethyllysine residues at Res-72 and Res-86, it is worthwhile to investigate protein methylase III from this source regarding its substrate specificity and physico-chemical properties. This aspect of research is in progress. BIOLOGICAL C’YTOCHROME
SIGNIFICANCE
OF
C METHYLATION
Earlier. Mitchell and his coworkers observed that both C, and C,, form of cytochrome c occurs in wildtype Nrurospora and the respiration-deficient mutants (Mitchell et al.. 1953). This would indicate that occurrence of two cytochrome c’s is not the result of the P&J mutation. A recent report of Verdiere & Lederer (1971) supports this contention; they have shown that two iso-cytochrome c’s synthesized by two strains of respiratory-deficient mutants of yeast (p-) contain one residue of c-N-trimethyllysine and that they do not differ in this respect from the iso-cytochrome c produced by p+ strain. Scott and Mitchell suggested that cytochrome c methylation would facilitate binding of this haemoprotein to the mitochondrial matrix (1969). This suggestion was mainly based on the observation that the pokq‘ strain of N. crassa accumulates up to 16 times the normal amount of cytochrome c found in the wild-type strain (Haskins er al., 1953) and that the only methylated form C, was extractable from the mitochondrial fraction (Scott & Mitchell, 1969). Recently, however. Pettigrew & Smith (1977) have suggested that cytochrome c methylation could be accidental, and not necessarily corresponding to a precise biological function. Indeed, as these authors have pointed out. lysine residue 72 (and residue 86
797
in plants) seems to be rather exposed to the solvent and accessible to an eventual methylation. However. in the view of the existence of highly cytochrome c-specific protein-lysine methyltransferase in N. crassa. the above hypothesis is highly teneous. One of the most ideal experimental conditions where one could investigate the biological significance of cytochrome c methylation appeared to methylate horse heart cytochrome c (since commercially available) with purified N. crassa protein-lysine methyltransferase, isolate the methylated cytochrome c and compare the methylated cytochrome c with unmethylated one, We have. however, encountered difficulties to methylate horse heart cytochrome c to any significant degree (Durban et cd., 1978); only 0.2’2 of the cytochrome c could by methylated. We are at loss to explain this. However, it is also possible that nascent cytochrome c is the genuine substrate for the enzyme, since one of the cyanogen bromide fragments of cytochrome c (residues l-65) incorporated the methyl group at approximately 12;:,. In order to overcome the above difficulty, we have prepared unmethylated and in uivo methylated iso-lcytochrome c’s from S. cvrrvisiae (Polastro et al., 1976) and compared these ftwo forms of cytochrome c, since these two cytochrome c’s have been shown to have identical amino acid sequence except at Res-72 where lysine is trimethylated (Foucher et al., 1972). By circular dichroism, redox potential and autooxidability kinetics measurement, it was shown that the haem environment and its coordination sphere is unaffected by the methylation state of the protein (Looze KTal.. 1976; Polastro rt a/.. 1976). Furthermore, those values found for horse heart cytochrome c were very close to those of yeast haemoprotein. In addition to the above, the thermal. acidic and guanidium hydrochloride denaturation of these two forms of yeast cytochrome c were also investigated (Polastro et al., 1976). as we could not find any significant difference between two cytochrome c’s Also. the circular dichroism spectra in the U.V. range was almost identical (Looze t’t al., 1976). These results indicate that the enzymatic methylation of cytochrome c at Res-72 did not affect the helical content, the structure nor the stability of the protein. The results are in accordance with the earlier observation that N. crassa cytochrome C, and C,, have identical sedimentation coefficients, and ultraviolet and visible absorption spectra (Scott & Mitchell, 1969). One of the hypothesis concerning the biological significance of protein methylation has been that methyl substitution of the e-NH, group of lysine protects the protein from the in civo proteolytic enzyme attack (Paik & Kim. 1975). Thus, the digestion kinetics of methylated and unmethylated cytochrome c by trypsin, yeast protease A and B were measured (Table 3). It can be seen that the “half digestion times” (T,.,) are almost unaffected by the methylation state of the haemoprotein. The possible objection that cytochrome c methylation in yeast could provide an effective protection only against the action of more specific protease( differing from the above result. can be easily ruled out by the work of Luzikov et al. (1976), who have shown that protease B. and protease A in a lesser degree, are responsible for the in
798
ENRICO POLASTRO
~7 ul.
Table 3. “Half digestion time” of horse heart and baker’s yeast (iso-I-methyiat~d and unmethylated~ cytochrome c with yeast protease A and B, and bovine trypsin (Polastro, unpublished results)
Half digestion time (T l/2) (mint* Proteolytic enzymes
Yeast iso- I xytochrome c Unmethylated Methylated
Trypsin (bovine) Protease B Protcase A
296.7 + 29.5 113.3f 17.0 >7 hr
300.0 k 12.2 115.0& 21.2 r7hr
Horse heart cytochrome c 160.0 * 8.2 360.0 * 16.3 >7hr
* “Half digestion time” has been defined as the length of time necessary to digest 50”,, of the haemo-
protein. t+rr>degradation of yeast cytochrome c. Conclusion that methylation of protein did not protect the protein from the proteolytic attack has also been shown with non-enzymatically methylated histones and pancreatic ribonuclease (Paik & Kim, 1972). Cytochrome c has been shown to be localized on the outer surface of the inner membrane of mitochondria (Dickerson & Timkovich, 1975). Thus, we have investigated the possible effect of enzymatic methylation of cytochrome e on its interaction and reactivity with mitochondria and some of the respiratory enzymes. The spectrophotometric determination of K, and relative V,,,_ value of the reaction of methylated and unmethylated yeast iso-I-cytochrome c with yeast sLlccinate dehydrogenase. cytochrome c peroxidase and cytochrome c oxidase failed to show any difference between the two forms of cytochrome c (Polastro cuta/.. 1977). However. the method employed relatively high ionic strength and cytochrome c concentration. thus masking an eventual difference between the two forms. No difference was also observed for the measurement of Ice characterizing the interaction of cytochrome c with cardiolip~n liposomes (Polastro. unpublished results). Next. we have studied the interaction of two forms c with cytochrome of yeast iso- 1-cytochrome c-depleted mitochondria (Polastro ef ~1.. 1978). As shown previously by others (Vanderkooi et ni.. 1973: Willi~~rns & Thorp. 1970; Ferguson-MilIer rr al.. 19761, this haemoprotein binds to the mit~hondfia by either strong and weak affinity binding sites (X, of 0.5 x lo- and lOmh M, respectively). It was found that the methylated form of yeast iso-1-cytochrome (’ interacts much more strongly vvith yeast mitochondria than the unmethylated cytochrome c in the high affinity zone (Fig. 1; panel B and C. Table 4). It can also be seen in Table 4 and Fig. 1 that the difference of K, is much less pronounced when heterologous mitochondrion from mammahan source was used. These results were obtained by three different independent methods: Oxygen consumption race (panel A) as described by Margoliash and his coworkers (1976). direct binding method (panel Bf and extrinsic fluorescence quenching (panel C). According to Margoliash et ai. (1976) the high athnity sites of cytochrome c to the mitochondria are provided by the cytochrome c oxidase, not by the mitochondrial membrane These authors also suggested that cytochrome c molecules in the high affinity binding sites function to complete the mitochondrial respiratory chain. From the above observations. it is concluded that methylation of Res-72 Iysine of cytochrome c could strengthen the interaction of the haemoprotein with
the cytochrome c onidase, in accordance with the earlier suggestion by ~kunuki et at. (196.5).This conclusion can also be further supported by a recent report by Ferguson-Miller t’r ul. (1978) and Smith c’t ai. (1977). who had shown that Res-72 lysine is iocated in the high affinity binding sites with the oxidase and that decrease of the positive charge associated with Res-72 lysine increased the Kn of the reaction. Indeed, trim~thyiation makes the c-NH2 group of lysine quaternary amine, thus making the charge permanent. regardless of the pH or the polarity condition. Margoliash 41 crl. (1977) have pointed out earlier that cytochrome c from different species react almost identically with their own oxidases (KD of 0.5 x JO-’ and 10wh MI. but very differentiy with the oxidases derived from other organisms. Thus, the observation that the Kr, of the methylated and unmethylated form of yeast iso-1-cytochrome c are almost identical when reacted with horse heart oxidase (lower panel of Fig. 1 and Table 4) could indicate that the oxidase could not recognize and discriminate between two forms of heterolo~ous yeast cytochrome c, as the yeast oxidase does. Therefore. methylation reaction of cytochrome c could be the consequence of evolutionary adaptation of this haemoprotein to the oxidase. The results presented in Fig. 1 and Table 4 could also be interpreted to mean that methylation of cytochrome c lowers the dissociation constant, thereby increasing the affinity towards the mitochondrial matrix. Since cytochrome c is synthesized outside of the mitochondria, the increase of its affinity towards the mitochondria will lower the amount of cytochrome c necessary to have a given quantity of bound and respiratorily active haemoprotein. This consequence will be extremely important for the physiology of yeast. particularly during the sudden transition of anaerobiosis to aerobiosis.
CONCLUDING REMARKS
The investigation on protein methylation can be divided into two stages. The first stage spans approximately 17 yr after the initial finding of the existence of r-N-methyllysine in the flagella protein of Saltt7ottella ryphirnuriwn in 1959. During this period, the scope of the protein methylation research has involved the investigation of: The kind of amino acid side chains methylated, the natural occurrence of in rice methylated protein, and the discovery and characterization of the specific enzymes involved. The year 1976 heralded the second stage of the research on
Cytochrome
c mcthylation
799
/ 0
I 2
I %
I 3
F.l.0.
Fig. I. Cytochrome c binding to cytochrome c-depleted yeast (top panel) or horse heart (fowcr panel) mitochondria. Cytochrome c-depleted mitochondr~a were prepared as previously described (Polastro et ui.. 1978). Cytochrome c binding was monitored by oxygen consumption rate (panel A). direct binding (panel B) or extrinsic fluorescence quanching (panel C). The assays were performed at 25 C in a 50mM MOPS (morphoiinopropane sulfonate) or cacodylate buffer at pH 7.2, containing 225 mM mannitol and 50mM sucrose. The following symbols are used; C&--O: Yeast iso-l-methylated cytochrome c: M: Unmethylated form and A-A: Horse heart cytochrome c. Panel A: Eadie Hofstee (Scatchard) representation of the oxygen consumption of cytochrome c-depleted mitochondria, incubated with increasing amounts of cytochrome c. Oxygen uptake was measured polarographicatly, using 1.4.N,N.N’.N’-tetramethylphenylenediamine (TMPD) as reducing agent, according to Ferguson-Miller et al. (1976). V, the oxygen consumption rate is expressed as nanomoles of oxygen consumed per minute in the assay and S is the cytochrome c concentration expressed as PM. Panel B: Scatchard plot of cytochrome c binding to cytochrome c-depleted mitochondria. incubated with increasing amounts of cytochrome c. After centrifugation. the concentration of free cytochrome c was determined spectrophotometrically in the supernatant. The amounts of bound cytochrome c was estimated in the pellet by the method described by Vanderkooi ct d. (1973). The following Scatchard equation is obtained: T/A = Kn - Kf. where K is the association constant (M -‘); F free cytochrome c concentration (FM): n the number of cytochrome c binding sites to the membrane (nmoles of cytochrome c per mg of protein); A the bound amount of cytochrome c (same units as n). Panel C: Scatchard plot of extrinsic fluorescence quenching of cytochrome c-depleted mitochondria by increasing amounts of cytochrome c. Fluorescent mitochondria were obtained by incubation wtth 10 PM of Dansyl phosspectra v+ere recorded phatidylethanolamine for 3 hr at U-2 C (Vanderkooi ef al.. 1973). Fluorescence with a Perkin-Elmer MPF 2A spectrofluoremeter. using 350 nm for excitation and emission. respectively. The addition of cytochrome c to the “fluorescent” mitochondrial suspension causes fluorescence quenching, lowering the quantum yield without altering the shape of the spectra. This suggests the quenching occurs mainly by an energy transfer mechanism. which is distance-dependent (Vanderkooi et al.. 1973). Consequently. we have assumed that fluorescence quenching was in relation with the amount of cytochrome c bound to the mitochondria. Since the amount of bound cytochrome c in this assay can not be directly determined, we assumed that this amount is extremely small thus ignored with regard to the analytical quantity of cytochrome c. The following Scatchard equation is obtained: ‘I,, F. 1. Q,‘cytochrome
c = K(“,, F. I. Q.) - KG
where K is the association constant (M-t): Ci a constant cytochrome c the analytical concentration of cytochrome of tluorescence intensity quenching. “ii F. I. Q. is calculated
where L and f are the fluorescence
9, F. I. Q. = 100 (1 - f&). intensity before and after (Polastro el Ul.. 1976).
methylation. This constitutes the study of the biological function of the reaction. In this review, we presented evidence which indicates that enzymatic methylation of Res-72 lysine of protein
related to the number of binding sites: e (j(M); and “#, F. I. Q.. the percentage by the equation cvtochrome
c addition.
respectively
cytochrome c in the Ascomycetes is to facilitate the binding of this haemoprotein to mitochondria. This experimental evidence is the second example showing the importance of protein methylation for the physi-
800 Table
ENRICO P~LASTR~ ur al. 4. Dissociation
constant
(K,, = I;K,)
Source of cytochrome c-depleted mitochondrla Yeast
Horse
heart
of cytochrome c interaction (Polastro c’t al.. 1978)
with
cytochrome
c,-depleted
Cqtochrome C’(,IM)* Yeast iso- I Methylated Unmethylated 0.049 (o.c.)** 0.04 k 0.0065 (f) 0.048 f 0.01 (d.b.) 0.078 (o.c.) 0.085 + 0.006 (f) 0.077 + 0.00X (d.b.)
Horse
0.104 (O.C.) 0. I2 f 0.005 (f) 0.11 f 0.014(d.h.l 0. I05 O.C.) 0. I I5 + 0.009 (f) 0.1 (d.b.)
mitochondria
heart
0.09 (0.C.) 0. I12 * 0.035 (f) 0.1 (d.b.1 0.097 (0.c ) 0.1 12 + 0.0015(f) 0.09s (d.b.1
* Assays were carried out in 0.225 M mannitol. 50 mM sucrose. and 50 mM MOPS buffer at pH 7.2 or cacodylatc buffer of pH 7.2 at 25 ‘C. Detailed experimental conditions are dcscrlbed under the legend of Fig. I. The values are the average of at least three independent experiments. each performed in quadruplicate. ** (o.c.): oxygen consumption rate. (0: fluorescence quenching method and (d.b.1: direct binding method. ologq of the organism. The first example was provided by LaBadie rt al. (1976) in 1976 who demonstrated protein
the precursor-product relationship between mcthylation and carnitine biosynthesis. Thus.
the investigation of the biological significance of protein methylation has just begun. and it is hoped that the next few years will bring a fruitful harvest in this highly diverse and complex post-translational covalent modification reaction. ,4cL~~o,~/~,tl~~~,~~~~~~t.s-TThis work was supported by research grants AM09602 from the National Institute of Arthritis. Metabolism and Digestive Diseases. CA10439 and CA12226 from the National Cancer Institute. and GM20594 from National Institute of General Medical Sciences of United States of America. Enrico Polastro is Aspirant of the Fonds National Beige de la Recherche Scientifique.
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tmtt1. 4ud.
Sci.. L’.S.,4. 73, 4050.
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31. I39
HASKINS F. A.. TISSIPRIX A.. MITCHELL H. K. & Mir(%n L.L M. B. (1953) Cytochromes and the succinic acid oxidase system of poky strain of !Vctr,o.~portr. J. hiol. Chm. 200. 819.
H~XZ~R G. (197.5) Tissue-specific blosynthesls ol’ t-Nmonomethyllysine and c-N-dimethyllysine in skeletal and cardiac muscle myosin: A model for the cell-free study of post-translational amino acid modification m proteins. J. UIO/UC Biol. 94, 3 11, KLEENE S. J.. TIERS M. L. 6t AI~LEK J. (1977) Isolation of glutamlc acid methyl ester from an Ewherichitr m/i membrane protein invol>cd in chemotauis. .I hiol C/IUU 252. 3214. LaBadie J. H.. Dunn W. A. & Aronson N. N. (1976) Hepatic synthesis of carnitine from protein-bound trimethylIysine. Biochrm. J. 1fXl X5. Loozt Y.. POLASTRO E.. GII-rtr\s C’. and Lroxis J. (1976) Iso-cytochromr (’ species from baker’s yeast (Analysis of their circular dichroism spectra). Biochou. J. 157. 773. Lur.~lcow V. N.. MAKI-IUS T. A & GAI UN A. V. (1976) A probable role of cell proteinases in the hiosqnthesis of mitochondrla in yeast. fERS Lcrt. 69. 108. MARGO;.IASH E. & S~HFITFR A. (1966) Cytochromc c. In 4dra~cr.s Protci?l Chrruistrv (edited bq ANFINSEN C. B.. ANSOX M. L.. F’I>SAL.IJ. T and RICHARI~S F. H.). Vol. 21. pp I Ii. Academic Press. New York
Cytochrome MARC;• LIASH E.. FFRCUOIV-MILL~.R S.. BRAU~GAN D. L.. KANG C. H. & D~THM~RS J. K. (1977) In Strucfurc~ UUC/ Funcfion qf Enrryy- Trunxluci!~y 1Me~t7hrunr.s(edited by VAN DAM K. & VAY GLLDER B. F.). Vol. 14. p. 69. Elsevier, Amsterdam. MITCHELL M. B.. MITCHTLL H. K. & TISSI~RI;S A. (1953) Mendelian and non-mendclian factors affecting the cytochrome system in Nrwmpor~r cra.s.w. Pux nutty. Jlcd. Sci.. U.S.A. 39. 606. MORSE R. K.. VIRC;ULS J. P.. MALLO\ J. & MCMANUS 1. R. (1975) Sitea of biological mcthqlation of proteins in cultured chick muscle cells. Biochc~77isrr~~ 14. 4316. NCKXLMSO’L S.. Dt KHAY E., KIM S & PAII( W. K. (1977) Cytochrome c.-specifc protein methqlase III from .I’wrospora
crussa.
Hfod7r/f7.
J.
165. I I
K.. W.An.% K.. MATS~ HARA H. & TAC~MOKI S. (1965)In O.uirlurion tu7d Rr/ufrd Rrdox Sy.stm. (edited by KING T. E.. MAX)\ H. S. & MORKISON M.). Vol. 2. p. 549. Wiley. New! York. PAIK W. K. & KIM S. (1970) Solubilization and partial purification of protein methylase III from calf thqmus nuclei. J. h/o/. Chut7. 245. 6010. PAIK W. K. & KIM S. (1972) Effect of mrthylation on susceptibility of protein to proteolytic enrymes. Riorhcnli\rry II. 2589. PAtK W. K. & KIM S. (1974) Role of histone-lysine methylation in cellular difierentiatlon. growth and development. In Post-S,~,nrhrtic Modljcurim oj Macro,,lo/ec,u/l,.~ (edited bj ANTONI F. & FARAGO A.). Vol. 34. p. 127. North-Holland.;American Elsevier. FEBS. Amsterdam. PAII< W. K. & KIM S. (1975) Protein methylation: ChemiCdl. enzjmological. and biological significance. In Adrrmcrs in Enzymlogy (edited by Mfxrr R A.). Vol. 42. pp. 227. Wiley. New York. PAII( W. K. & KIM. S. (1977) Carnitine biosynthesis via protein methylatlon. TIBS 2. 159. P~-~TIc+RE~ G. W. & Shun! G. M. (I 9771 Novel N-terminal
OKLINUI(I
R‘
917
”
(’ methqlation protein blocking Natlrrc 265. 661.
801 group
idcntlficd
as dimethylproline
POLASTRO E.. Looozr. Y. & LLONIS J. (1976) Study of the biological significance of cytochromc methylation. Riochi/,l. hioph~u. Acru 446. 3 IO. POLASTR~ E.. Loozt Y. & LI ONIS J. (1977) Biological significance of methylatlon of cytochromcs from .~.\u~uI~~(‘Ic.\ and plants. P/t?,/l)~h[,~rir.\rr~ 16. 639. POLASTRO E., Dt CONINCR M. M.. D~.voc;t L M. R.. MAILII R E. L.. LOOZI Y.. SrHxt K A. G. & LroNIs J. (197X) Evidencc that trimethylation of iso-I-cytochromc c from Suc( huro~7~w~c wrwi.ucrc affects interaction with the mitochondrla. FEBS Lcrr. 86. 17. RAMSHAW J. A. M. & BoLLTI R D. (lY75) Amino acid cequence of q tochromc (’ from mgcr-seed, Guizoritr ub~~ \irlrcu.P/7~/oc/7c,,,7rstr~ 14. 1945. SCOTT W. A. 8: MITCH~LI. H. K. (1969) Secondary modifcation of cytochrome (’ by Ncrrrovporrr uuuu. B1oclte177i.\1rj 8. 4282. SMlrH H. T., STALJIXNMAYIK N. & MtLLlrr F. (1977) Use of specific lysine modilications to locate the reaction site of cytochromc c wtth cytochrome oxidase. H/oc/n,nri.\tr~ 16. 497 I. VANI)I RUMX J.. EKI-CIN%A M. & CHANC r B. (1973) Cytochrome (’ interaction with membranes. 1. USC of a tluorascent chromophore in the study of cytochrome c interaction with artificial and mitochondrial membranes. Arch Biochw77. Biop/7jx 154. 2 19. VI.RIXI RI. J. & Lr 7x wR F. (1971) Prcsencc de la c-N-trimethyllys7nc dans L‘iso-I et L’ISO-2 cytochromc c synthetises par des souches de Icvurc a deficlonce rcspnatoire (tj-). FEBS Lrrr. 19. 71. WILLIAMS J. N. & THORP S. L. (1970) lnfluencc of dcgradative procedures. salts. respiratory Inhibitors. and gramicidin on the bmding of cytochrome (’ by liver mitochondria. .4r(h.t Riochw~. Bioph~‘.t. 141. 622.
C METHYLATION
ENRICO POLASTRO*, ARTHUR G. SCIHNECK*, JOSE LEONIS*. SANGDUK KIM and WOON
Kr PAIK
Chimie
Generale 1. Faculte des Sciences, Universite Libre de Bruxelles. Belgique*. and Fels Research Institute and Department of Biochemistry, Temple University School of Medicine. Philadelphia, PA. 19140, U.S.A. (Receioed 29 March 1978)
Abstract-Enzymatic methylation of 72 lysyl residue of cytochrome c in lower organisms facilitates its binding to mitochondria, and subsequently plays an important role in the electron transport process.
Protein methylation is an ubiquitously occurring post-translational modification reaction involving lysine, arginine, dicarboxylic amino acids and histidine side chains (Paik & Kim, 1975). Biochemical significance of the reaction is not generally well understood. It is however being actively investigated in the area of bacterial chemotaxis (Kleene et al., 1977; Werf & Koshland, 1977), processing of pituitary hormones (Diliberto & Axelrod, 1974), exocytosis (Diliberto et al.. 1976), gene regulation (Byvoet & Baxter, 1975; Paik & Kim, 1974), and muscle contraction (Morse et al.. 1975; Huszar, 1975). Recent evidence strongly indicates that carnitine, which is an acyl carrier within the mitochondrial membrane, is synthesized from l-N-trimethyllysine and that the formation of E-N-trimethyllysine results from enzymatic methylation of protein-lysine residues using S-adenosyl-L-methionine and subsequent degradation by proteolytic enzymes (LaBadie et al., 1976; Paik & Kim, 1977). In this review, we present evidence which indicates that enzymatic trimethylation of cytochrome c of yeast or Neurospora crassa facilitates the binding of the cytochrome c with mitochondria, and subsequently play an important role in the process of electron transport. HISTORICAL BACKGROUND Cytochrome
c is a universally
occurring
protein,
found
in the mitochondrion of all eukaryotes. Because of its universality and ease of preparation, many studies have been devoted to its primary and tertiary structure. In fact. cytochrome c is one of the proteins most thoroughly studied. and cytochrome c from up to 100 species of microorganisms, plants, and animals have been completely sequenced (Dayhoff et al., 1972). Since the biological role of cytochrome c remained the same throughout evolution. one would expect that the primary structure of the protein from various sources should not differ too far from each other. However, it was found that the cytochrome c in man has diverged from the cytochrome c in Neurospora crassa in 44 out of 104 places (DeLange et al., 1969), yet approximately 34 amino acid residues remaining invariant. Among the invariant amino acid residues, 795
the undecapeptide of residues 70 and 80 is the largest sequence in all the cytochrome c (Margoliash & Schejter, 1966). Thus, this peptide seems to be of prime importance for the function of the protein, any mutation in it being probably lethal. In 1969, DeLange et al. demonstrated that the 70-80 undecapeptide of the cytochrome c is not absolutely invariant in all species: Cytochrome c of wheat germ and Neurospora crussa were found to contain c-N-trimethyllysine residue in place of lysine at Res-72 position (1969). Since then, the cytochrome ( of many other organisms also were found to contain this residue; for example, Neurosporu c~u.w~. Succharomyces crrecisiae. and Cundida krusei contain a single C-N-trimethyllysine at Res-72, while wheat germ, Spinacea oleracea (spinach) and AIltmm porrutn (leak) contain 2 e-N-trimethyllysine at Res-72 and Res-86 (Brown & Boulter, 1973; Brown et al.. 1973; DeLange et al., 1970; Ramshaw & Boulter, 1975). However, the following organisms contain no methylated lysine: Lamprey, dogfish, bullfrog, turtle, rattlesnake, turkey. mammals and insect (DeLange et al.. 1970). These findings indicate a sharp evolutionary differentiation in the development of specific methylation enzymes among Ascotnycetes, higher plants, vertebrates and insects. In N. crassa, there are two species of cytochrome c, namely C, and C,,, differing only at the stage of residues 72, this position being occupied by a E-N-trimethyllysine in C, and a lysine in C,, (Scott & Mitchell. 1969). Working with N. crassa poky mutant (mi-l), Scott and Mitchell demonstrated by the use of pulse-chase experiment that cytochrome C,, form was first formed and then converted to C, on ageing (Scott & Mitchell, 1969). This observation strongly suggested that methylation of the c-amino group of lysine residue occurs on the whole cytochrome c polypeptide level. In contrast to the case of N. crassa. in Saccharomyces cereuisiae. where the unmethylated form of cytochrome c coexists in vic’o with the methylated form (Foucher et al., 1972). Brunko could not find any evidence of in bico conversion of the unmethylated cytochrome c to the methylated (Brunko. 1972). He has consequently suggested that cyto-
ENKIC~ POLASTKO cf ul.
796 Table
I. Substrate
Source of cytochromc Horse Rabbit Beef Guanaco Mouse Pigeon Chicken Tuna
activity
of various
cytochromes
c for protein-lysine
Moles of t-N-trimethyllysinc mole of cytochrome c irr ~~ilo
(’
(Llama)
to 1.26 nmole
of [methyl-‘4C]
I oo*
I I? II? X6 57
I20 66 96 40 166 -I incorporated/min/mg
ENZYMOLOGY
In order to investigate this specific methylation of lysine Res-72 in the cytochrome c, a protein methylase III [S-Adenosylmethionine:protein-lysine methyltransferase; EC 2. I. I.431 was recently purified from N. crassa approximately 3500-fold (Nochumson YI al.. 1977: Durban et al.. 1978). The enzyme is highly specific in its protein substrate requirement; of all proteins tested. only cytochrome c had substrate activity. Cytochrome c from various species were able to act as methyl accepting protein substrates in a range of 5@150”, compared to horse heart cytochrome c (Table I). Only one cytochrome c species was a very poor substrate, iso-l of baker’s yeast. This finding supports the notion that only unmethylated cytochrome c can act as methyl acceptor and once position 72 is methylated. the protein-lysine methyltransferase can not add any further methyl groups, The purified protein methylase III of N. Crassa appears to recognize the amino acid sequence of -XLys-Lys-Y- where X and Y may vary but a double lysine sequence is of absolute necessity (Durban er al., 1978). Even though I9 lysine residues are present in 104 amino acid residues of horse heart cytochrome
* N.A. represents
tion
site at lysine
not active
protein.
residues
7 8 not
of horse heart cytochrome (Durban ct al.. 1978)
K, (mM)
Cytochrome c (horse heart): Native Ethanol-denatured Heme-free CNBr-peptide: Peptide I (residues l-80) Peptide II (residues l-65) Peptide III (residues 666104) Peptide IV (residues 81-104) Peptide V (residues 66-80)
of enzyme
c, only single lysine residue at position 72 is methylated, which coincides with the lysine found methylated irl r:ica in cytochrome c of N. crassa. Horse heart cytochrome c contains 2 methionyl residues at position 65 and 80; however, treatment of the protein with cyanogen bromide in 0.1 N HCI yielded five peptides due to incomplete reaction. consisting of residues I-80. residues l-65. residues 66-104. residues 8l-- 104, and residues 6680 (peptides I, II. III. IV and V, respectively (Durban et al., 1978). Peptide I. II and III were good substrate for the enzyme. having low K, values. while peptide IV and V were completely inactive as methyl-accepting substrate (Table 2). The results strongly indicate that not only the reactive site such as -X-Lys-Lys-Y- sequence is necessary, but also the length of the polypeptide chain is an important feature in determining methylaccepting activity. When present in the intact molecule of horse heart cytochrome c, peptide 11 (residues l-65) cannot be methylated with S-adenosyl-L-methionine by the purified protein-lysine methyltransferase (protein methylase III) (Durban et ul.. 1978). However. the peptide served as an excellent substrate for the enzyme (with high V,,, and low K, value; Table 2) and further studies indicated that chymotryptic peptide containing residue 7 lysine (-X-Lys.‘7-Lys/8-Y-) was the only one methylated. The reason for the potential methyla-
Parameters of various CNBr-peptides protein-lysine methyltransferase
tested
methvl accepting activ;ty
0
chrome c methylation in S. crrrcisiuc~ occurs on the nascent polypeptide stage, analogous to the enzymatic methylation of myosin (Morse er a/.. 1975).
2. Kinetic
of iz’. c’r’uss~r
0 0 0 0 0 0 0 0
* Corresponds
Substrate
Relative
0
Frog Silkworm Baker’s yeast
Table
methyltransferasc
0.32 _+ 0.13 0.20 0.03 0.007 0.04 0.04 N.A.* N.A.
being
methylated
c for !V. cr~z.w
V ,“.I\ (nmole methyl~min;mg emyme protein)
‘4 _+ h 29 7 I6 27 2.4 N.A. N.A.
Cytochrome c methylation in intact cytochrome c, particularly “native” one, may be due to the fact that if the enzyme binds its protein substrate at the “front” orientation of the molecule, the lysine residues 778 are positioned in back essentially hidden from the enzyme (Durban rt al.. 1978). Thus, in peptide fragment II, the constraints imposed on lysine residue 7-8 as part of native cytochrome c are removed and this methylation site now becomes freely accessible to the enzyme for methylation. Contrary to the irr oivo product, the methylation products of horse heart cytochrome c by the purified N. crassa protein-lysine methyltransferase showed three methylated amino acids, i.e. E-N-monomethyllysine, l-N-dimethyllysine and l-N-trimethyllysine in a ratio of 1:3:4 (Durban er al., 1978). This ratio remained constant throughout 3500-fold purification, suggesting that a single enzyme is responsible for methylation of all three lysines by stepwise mechanism. The optimum pH of the enzyme is 9.0, its pl is 4.8, and its molecular weight is about 120,000. The K, value for cytochrome c is 0.3 mM. the K, for S-adenosyl-t.-methionine is 19 PM, while the inhibition constant K, for S-adenosyl-t_-homocysteine is about 2pM. It is also of interest to note that histone did not accept methyl groups from S-adenosyl-L-methionine with the N. crmsu enzyme (Durban rt a/.. 1978). This is in sharp contrast to the enzyme from calf thymus or rat brain which preferentially acts on histones (Paik & Kim. 1970; Duerre et al.. 1977). while cytochrome c is a poor substrate. Since wheat germ cytochrome c contains two l-N-trimethyllysine residues at Res-72 and Res-86, it is worthwhile to investigate protein methylase III from this source regarding its substrate specificity and physico-chemical properties. This aspect of research is in progress. BIOLOGICAL C’YTOCHROME
SIGNIFICANCE
OF
C METHYLATION
Earlier. Mitchell and his coworkers observed that both C, and C,, form of cytochrome c occurs in wildtype Nrurospora and the respiration-deficient mutants (Mitchell et al.. 1953). This would indicate that occurrence of two cytochrome c’s is not the result of the P&J mutation. A recent report of Verdiere & Lederer (1971) supports this contention; they have shown that two iso-cytochrome c’s synthesized by two strains of respiratory-deficient mutants of yeast (p-) contain one residue of c-N-trimethyllysine and that they do not differ in this respect from the iso-cytochrome c produced by p+ strain. Scott and Mitchell suggested that cytochrome c methylation would facilitate binding of this haemoprotein to the mitochondrial matrix (1969). This suggestion was mainly based on the observation that the pokq‘ strain of N. crassa accumulates up to 16 times the normal amount of cytochrome c found in the wild-type strain (Haskins er al., 1953) and that the only methylated form C, was extractable from the mitochondrial fraction (Scott & Mitchell, 1969). Recently, however. Pettigrew & Smith (1977) have suggested that cytochrome c methylation could be accidental, and not necessarily corresponding to a precise biological function. Indeed, as these authors have pointed out. lysine residue 72 (and residue 86
797
in plants) seems to be rather exposed to the solvent and accessible to an eventual methylation. However. in the view of the existence of highly cytochrome c-specific protein-lysine methyltransferase in N. crassa. the above hypothesis is highly teneous. One of the most ideal experimental conditions where one could investigate the biological significance of cytochrome c methylation appeared to methylate horse heart cytochrome c (since commercially available) with purified N. crassa protein-lysine methyltransferase, isolate the methylated cytochrome c and compare the methylated cytochrome c with unmethylated one, We have. however, encountered difficulties to methylate horse heart cytochrome c to any significant degree (Durban et cd., 1978); only 0.2’2 of the cytochrome c could by methylated. We are at loss to explain this. However, it is also possible that nascent cytochrome c is the genuine substrate for the enzyme, since one of the cyanogen bromide fragments of cytochrome c (residues l-65) incorporated the methyl group at approximately 12;:,. In order to overcome the above difficulty, we have prepared unmethylated and in uivo methylated iso-lcytochrome c’s from S. cvrrvisiae (Polastro et al., 1976) and compared these ftwo forms of cytochrome c, since these two cytochrome c’s have been shown to have identical amino acid sequence except at Res-72 where lysine is trimethylated (Foucher et al., 1972). By circular dichroism, redox potential and autooxidability kinetics measurement, it was shown that the haem environment and its coordination sphere is unaffected by the methylation state of the protein (Looze KTal.. 1976; Polastro rt a/.. 1976). Furthermore, those values found for horse heart cytochrome c were very close to those of yeast haemoprotein. In addition to the above, the thermal. acidic and guanidium hydrochloride denaturation of these two forms of yeast cytochrome c were also investigated (Polastro et al., 1976). as we could not find any significant difference between two cytochrome c’s Also. the circular dichroism spectra in the U.V. range was almost identical (Looze t’t al., 1976). These results indicate that the enzymatic methylation of cytochrome c at Res-72 did not affect the helical content, the structure nor the stability of the protein. The results are in accordance with the earlier observation that N. crassa cytochrome C, and C,, have identical sedimentation coefficients, and ultraviolet and visible absorption spectra (Scott & Mitchell, 1969). One of the hypothesis concerning the biological significance of protein methylation has been that methyl substitution of the e-NH, group of lysine protects the protein from the in civo proteolytic enzyme attack (Paik & Kim. 1975). Thus, the digestion kinetics of methylated and unmethylated cytochrome c by trypsin, yeast protease A and B were measured (Table 3). It can be seen that the “half digestion times” (T,.,) are almost unaffected by the methylation state of the haemoprotein. The possible objection that cytochrome c methylation in yeast could provide an effective protection only against the action of more specific protease( differing from the above result. can be easily ruled out by the work of Luzikov et al. (1976), who have shown that protease B. and protease A in a lesser degree, are responsible for the in
798
ENRICO POLASTRO
~7 ul.
Table 3. “Half digestion time” of horse heart and baker’s yeast (iso-I-methyiat~d and unmethylated~ cytochrome c with yeast protease A and B, and bovine trypsin (Polastro, unpublished results)
Half digestion time (T l/2) (mint* Proteolytic enzymes
Yeast iso- I xytochrome c Unmethylated Methylated
Trypsin (bovine) Protease B Protcase A
296.7 + 29.5 113.3f 17.0 >7 hr
300.0 k 12.2 115.0& 21.2 r7hr
Horse heart cytochrome c 160.0 * 8.2 360.0 * 16.3 >7hr
* “Half digestion time” has been defined as the length of time necessary to digest 50”,, of the haemo-
protein. t+rr>degradation of yeast cytochrome c. Conclusion that methylation of protein did not protect the protein from the proteolytic attack has also been shown with non-enzymatically methylated histones and pancreatic ribonuclease (Paik & Kim, 1972). Cytochrome c has been shown to be localized on the outer surface of the inner membrane of mitochondria (Dickerson & Timkovich, 1975). Thus, we have investigated the possible effect of enzymatic methylation of cytochrome e on its interaction and reactivity with mitochondria and some of the respiratory enzymes. The spectrophotometric determination of K, and relative V,,,_ value of the reaction of methylated and unmethylated yeast iso-I-cytochrome c with yeast sLlccinate dehydrogenase. cytochrome c peroxidase and cytochrome c oxidase failed to show any difference between the two forms of cytochrome c (Polastro cuta/.. 1977). However. the method employed relatively high ionic strength and cytochrome c concentration. thus masking an eventual difference between the two forms. No difference was also observed for the measurement of Ice characterizing the interaction of cytochrome c with cardiolip~n liposomes (Polastro. unpublished results). Next. we have studied the interaction of two forms c with cytochrome of yeast iso- 1-cytochrome c-depleted mitochondria (Polastro ef ~1.. 1978). As shown previously by others (Vanderkooi et ni.. 1973: Willi~~rns & Thorp. 1970; Ferguson-MilIer rr al.. 19761, this haemoprotein binds to the mit~hondfia by either strong and weak affinity binding sites (X, of 0.5 x lo- and lOmh M, respectively). It was found that the methylated form of yeast iso-1-cytochrome (’ interacts much more strongly vvith yeast mitochondria than the unmethylated cytochrome c in the high affinity zone (Fig. 1; panel B and C. Table 4). It can also be seen in Table 4 and Fig. 1 that the difference of K, is much less pronounced when heterologous mitochondrion from mammahan source was used. These results were obtained by three different independent methods: Oxygen consumption race (panel A) as described by Margoliash and his coworkers (1976). direct binding method (panel Bf and extrinsic fluorescence quenching (panel C). According to Margoliash et ai. (1976) the high athnity sites of cytochrome c to the mitochondria are provided by the cytochrome c oxidase, not by the mitochondrial membrane These authors also suggested that cytochrome c molecules in the high affinity binding sites function to complete the mitochondrial respiratory chain. From the above observations. it is concluded that methylation of Res-72 Iysine of cytochrome c could strengthen the interaction of the haemoprotein with
the cytochrome c onidase, in accordance with the earlier suggestion by ~kunuki et at. (196.5).This conclusion can also be further supported by a recent report by Ferguson-Miller t’r ul. (1978) and Smith c’t ai. (1977). who had shown that Res-72 lysine is iocated in the high affinity binding sites with the oxidase and that decrease of the positive charge associated with Res-72 lysine increased the Kn of the reaction. Indeed, trim~thyiation makes the c-NH2 group of lysine quaternary amine, thus making the charge permanent. regardless of the pH or the polarity condition. Margoliash 41 crl. (1977) have pointed out earlier that cytochrome c from different species react almost identically with their own oxidases (KD of 0.5 x JO-’ and 10wh MI. but very differentiy with the oxidases derived from other organisms. Thus, the observation that the Kr, of the methylated and unmethylated form of yeast iso-1-cytochrome c are almost identical when reacted with horse heart oxidase (lower panel of Fig. 1 and Table 4) could indicate that the oxidase could not recognize and discriminate between two forms of heterolo~ous yeast cytochrome c, as the yeast oxidase does. Therefore. methylation reaction of cytochrome c could be the consequence of evolutionary adaptation of this haemoprotein to the oxidase. The results presented in Fig. 1 and Table 4 could also be interpreted to mean that methylation of cytochrome c lowers the dissociation constant, thereby increasing the affinity towards the mitochondrial matrix. Since cytochrome c is synthesized outside of the mitochondria, the increase of its affinity towards the mitochondria will lower the amount of cytochrome c necessary to have a given quantity of bound and respiratorily active haemoprotein. This consequence will be extremely important for the physiology of yeast. particularly during the sudden transition of anaerobiosis to aerobiosis.
CONCLUDING REMARKS
The investigation on protein methylation can be divided into two stages. The first stage spans approximately 17 yr after the initial finding of the existence of r-N-methyllysine in the flagella protein of Saltt7ottella ryphirnuriwn in 1959. During this period, the scope of the protein methylation research has involved the investigation of: The kind of amino acid side chains methylated, the natural occurrence of in rice methylated protein, and the discovery and characterization of the specific enzymes involved. The year 1976 heralded the second stage of the research on
Cytochrome
c mcthylation
799
/ 0
I 2
I %
I 3
F.l.0.
Fig. I. Cytochrome c binding to cytochrome c-depleted yeast (top panel) or horse heart (fowcr panel) mitochondria. Cytochrome c-depleted mitochondr~a were prepared as previously described (Polastro et ui.. 1978). Cytochrome c binding was monitored by oxygen consumption rate (panel A). direct binding (panel B) or extrinsic fluorescence quanching (panel C). The assays were performed at 25 C in a 50mM MOPS (morphoiinopropane sulfonate) or cacodylate buffer at pH 7.2, containing 225 mM mannitol and 50mM sucrose. The following symbols are used; C&--O: Yeast iso-l-methylated cytochrome c: M: Unmethylated form and A-A: Horse heart cytochrome c. Panel A: Eadie Hofstee (Scatchard) representation of the oxygen consumption of cytochrome c-depleted mitochondria, incubated with increasing amounts of cytochrome c. Oxygen uptake was measured polarographicatly, using 1.4.N,N.N’.N’-tetramethylphenylenediamine (TMPD) as reducing agent, according to Ferguson-Miller et al. (1976). V, the oxygen consumption rate is expressed as nanomoles of oxygen consumed per minute in the assay and S is the cytochrome c concentration expressed as PM. Panel B: Scatchard plot of cytochrome c binding to cytochrome c-depleted mitochondria. incubated with increasing amounts of cytochrome c. After centrifugation. the concentration of free cytochrome c was determined spectrophotometrically in the supernatant. The amounts of bound cytochrome c was estimated in the pellet by the method described by Vanderkooi ct d. (1973). The following Scatchard equation is obtained: T/A = Kn - Kf. where K is the association constant (M -‘); F free cytochrome c concentration (FM): n the number of cytochrome c binding sites to the membrane (nmoles of cytochrome c per mg of protein); A the bound amount of cytochrome c (same units as n). Panel C: Scatchard plot of extrinsic fluorescence quenching of cytochrome c-depleted mitochondria by increasing amounts of cytochrome c. Fluorescent mitochondria were obtained by incubation wtth 10 PM of Dansyl phosspectra v+ere recorded phatidylethanolamine for 3 hr at U-2 C (Vanderkooi ef al.. 1973). Fluorescence with a Perkin-Elmer MPF 2A spectrofluoremeter. using 350 nm for excitation and emission. respectively. The addition of cytochrome c to the “fluorescent” mitochondrial suspension causes fluorescence quenching, lowering the quantum yield without altering the shape of the spectra. This suggests the quenching occurs mainly by an energy transfer mechanism. which is distance-dependent (Vanderkooi et al.. 1973). Consequently. we have assumed that fluorescence quenching was in relation with the amount of cytochrome c bound to the mitochondria. Since the amount of bound cytochrome c in this assay can not be directly determined, we assumed that this amount is extremely small thus ignored with regard to the analytical quantity of cytochrome c. The following Scatchard equation is obtained: ‘I,, F. 1. Q,‘cytochrome
c = K(“,, F. I. Q.) - KG
where K is the association constant (M-t): Ci a constant cytochrome c the analytical concentration of cytochrome of tluorescence intensity quenching. “ii F. I. Q. is calculated
where L and f are the fluorescence
9, F. I. Q. = 100 (1 - f&). intensity before and after (Polastro el Ul.. 1976).
methylation. This constitutes the study of the biological function of the reaction. In this review, we presented evidence which indicates that enzymatic methylation of Res-72 lysine of protein
related to the number of binding sites: e (j(M); and “#, F. I. Q.. the percentage by the equation cvtochrome
c addition.
respectively
cytochrome c in the Ascomycetes is to facilitate the binding of this haemoprotein to mitochondria. This experimental evidence is the second example showing the importance of protein methylation for the physi-
800 Table
ENRICO P~LASTR~ ur al. 4. Dissociation
constant
(K,, = I;K,)
Source of cytochrome c-depleted mitochondrla Yeast
Horse
heart
of cytochrome c interaction (Polastro c’t al.. 1978)
with
cytochrome
c,-depleted
Cqtochrome C’(,IM)* Yeast iso- I Methylated Unmethylated 0.049 (o.c.)** 0.04 k 0.0065 (f) 0.048 f 0.01 (d.b.) 0.078 (o.c.) 0.085 + 0.006 (f) 0.077 + 0.00X (d.b.)
Horse
0.104 (O.C.) 0. I2 f 0.005 (f) 0.11 f 0.014(d.h.l 0. I05 O.C.) 0. I I5 + 0.009 (f) 0.1 (d.b.)
mitochondria
heart
0.09 (0.C.) 0. I12 * 0.035 (f) 0.1 (d.b.1 0.097 (0.c ) 0.1 12 + 0.0015(f) 0.09s (d.b.1
* Assays were carried out in 0.225 M mannitol. 50 mM sucrose. and 50 mM MOPS buffer at pH 7.2 or cacodylatc buffer of pH 7.2 at 25 ‘C. Detailed experimental conditions are dcscrlbed under the legend of Fig. I. The values are the average of at least three independent experiments. each performed in quadruplicate. ** (o.c.): oxygen consumption rate. (0: fluorescence quenching method and (d.b.1: direct binding method. ologq of the organism. The first example was provided by LaBadie rt al. (1976) in 1976 who demonstrated protein
the precursor-product relationship between mcthylation and carnitine biosynthesis. Thus.
the investigation of the biological significance of protein methylation has just begun. and it is hoped that the next few years will bring a fruitful harvest in this highly diverse and complex post-translational covalent modification reaction. ,4cL~~o,~/~,tl~~~,~~~~~~t.s-TThis work was supported by research grants AM09602 from the National Institute of Arthritis. Metabolism and Digestive Diseases. CA10439 and CA12226 from the National Cancer Institute. and GM20594 from National Institute of General Medical Sciences of United States of America. Enrico Polastro is Aspirant of the Fonds National Beige de la Recherche Scientifique.
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OKLINUI(I
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801 group
idcntlficd
as dimethylproline
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