Cytochrome c methyltransferase, Ctm1p, of yeast

Cytochromes c from plants and fungi, but not higher animals, contain methylated lysine residues at specific positions, including for example, the trimethylated lysine at position 72 in iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. Testing of 6,144 strains of S. cerevisiae, each overproducing a different open reading frame fused to glutathione S-transferase, previously revealed that YHR109w was associated with an activity that methylated horse cytochrome c. We show here that this open reading frame, denoted Ctm1p, is specifically responsible for trimethylating lysine 72 of iso-1-cytochrome c. Unmethylated forms of cytochrome c but not other proteins or nucleic acids are methylated in vitro by Ctm1p produced in S. cerevisiae or Escherichia coli. Iso-1-cytochrome c purified from a ctm1-Delta strain is not trimethylated in vivo, whereas the K72R mutant form, or the trimethylated Lys-72 form of iso-1-cytochrome c, are not significantly methylated by Ctm1p in vitro. Like apocytochrome c, but in contrast to holocytochrome c, Ctm lp is located in the cytosol, consistent with the view that the natural substrate is apocytochrome c. The ctm1-Delta strain lacking the methyltransferase did not exhibit any growth defect on a variety of media and growth conditions, and the unmethylated iso-1-cytochrome c was produced at the normal level and exhibited the normal activity in vivo. Ctm1p and cytochrome c were coordinately regulated during anaerobic to aerobic transition, a finding consistent with the view that this methyltransferase evolved to act on cytochrome c.     

Effect of enzymatic methylation of apocytochrome c on holocytochrome c formation and proteolysis

1. Methylation of the lysine at residue 72 of yeast apocytochrome c increases its import into mitochondria. 2. Using methylated and unmethylated apocytochrome c as substrate and intact yeast mitochondria and a solubilized mitochondrial fraction as a source of cytochrome c heme lyase, the results show that the methylation state of the apoprotein has no significant effect on its conversion to holoprotein. 3. The above result suggests that the import mechanism is separate from the heme-attaching activity. 4. Unmethylated apocytochrome c was less resistant to a yeast homogenate fraction that methylated apocytochrome c, suggesting that methylation of apocytochrome c alters the conformation of the whole protein.     

Cytochrome c methylation

In this review, protein methylation is outlined in general terms, highlighting the major amino acids that are methylated and some of the proteins in which they are found. The majority of the review examines the methylation of cytochrome c at Lys-77 of lower eukaryotes as a possible model for methylation studies. Early work involving the purification and characterization of the methyltransferase responsible for this methylation indicated cytochrome c was methylated posttranslationally, yet prior to import into the mitochondria. Methylation in vitro occurred only at the in vivo methylation site and only on cytochrome c. Later studies using in vitro translated apocytochrome c revealed that methylated, as compared with unmethylated, apocytochrome c was imported preferentially into yeast, but not rat liver, mitochondria. Efforts to discover the reasons for this preference have shown that methylation of apocytochrome c dramatically lowers its isoelectric point (against a predicted increase) and decrease its Stokes radius. A possible mechanism for these differences involving the disruption of hydrogen bonds is presented here with space-filling models. Finally, the in vivo significance of this modification is also discussed.     

Study of the biological significance of cytochrome methylation. I. Thermal, acid and guanidinium hydrochloride denaturations of baker's yeast ferricytochromes c.

The iso-cytochromes c from baker's yeast: iso-1 methylated and unmethylated forms and iso-2 have been purified and their stabilities towards denaturants compared to that of horse heart cytochrome c. Thermal, acid and guanidinium hydrochloride denaturations were followed using fluorescence emission of their tryptophan 59 and/or the absorbance in the Soret region as the physical parameters. Very few differences could be evidenced among the ferricytochromes investigated in this study insofar as the acid denaturations are concerned. This is to be contrasted with the conclusions of the thermal and guanidinium hydrochloride denaturations studies which clearly showed the ferricytochrome from horse heart to be much more stable than those from baker's yeast. No appreciable differences could be measured among the methylated and unmethylated forms of iso-1 cytochrome c nor among iso-1 and iso-2 cytochromes from baker's yeast. Our results suggest that a stabilizing effect of methylation on the tridimensional structure of ferricytochrome c must probably be discarded. Other possible physiological roles of methylation are suggested taking into account the relative instability of ascomycetes's cytochromes as compared to mammalian ones.     

Enzymatic trimethylation of lysine-72 in cytochrome c

The present observations are the continuation of our earlier study on the physicochemical mechanism of protein-lysine methylation. In this paper the electrophoretic behaviour (pI values) of two chemically modified horse heart cytochromes c at lysine-72 with trifluoromethylphenylcarbamoyl (neutral group) or carboxydinitrophenyl (acidic group) is compared with the enzymatically methylated cytochrome c. The results indicate that although both chemically modified cytochromes c have lower pI values than the unmodified cytochrome c, the enzymatic methylation appears to be much more efficient in lowering the pI values of the protein than the chemical modification. Furthermore, the lowering of the pI value of cytochrome c by enzymatic methylation is highly dependent on the urea concentration. The presence of urea reduces the effect of methylation on the protein molecule and the difference in pI values virtually disappears with the increasing concentration of urea (6 M), which essentially disrupts the protein tertiary structure.     

Biogenesis of cytochrome c in Neurospora crassa. Synthesis of apocytochrome c, transfer to mitochondria and conversion to Holocytochrome c.

1. Precipitating antibodies specific for apocytochrome c and holocytochrome c, respectively, were employed to study synthesis and intracellular transport of cytochrome c in Neurospora in vitro. 2. Apocytochrome c as well as holocytochrome c were found to be synthesized in a cell-free homogenate. A precursor product relationship between the two components is suggested by kinetic experiments. 3. Apocytochrome c synthesized in vitro was found in the post-ribosomal fraction and not in the mitochondrial fraction, whereas holocytochrome c synthesized in vitro was mainly detected in the mitochondrial fraction. A precursor product relationship between postribosomal apocytochrome c and mitochondrial holocytochrome c is indicated by the labelling data. In the microsomal fraction both apocytochrome c and holocytochrome c were found in low amounts. Their labeling kinetics do not subbest a precursor role of microsomal apocytochrome c or holocytochrome c. 4. Formation of holocytochrome c from apocytochrome c was observed when postribosomal supernatant containing apocytochrome c synthesized in vitro was incubated with isolated mitochondria, but not when incubated in the absence of mitochondria. The cytochrome c formed under these conditions was detected in the mitochondria. 5. Conversion of labelled apocytochrome c synthesized in vitro to holocytochrome c during incubation of a postribosomal supernatant with isolated mitochondria was inhibited when excess isolated apocytochrome c, but not when holocytochrome c was added. 6. The data presented are interpreted to show that apocytochrome c is synthesized on cytoplasmic ribosomes and released into the supernatant. It is suggested that apocytochrome c migrates to the inner mitochondrial membrane, where the heme group is covalently linked to the apoprotein. The hypothesis is put forward that the concomitant change in conformation leads to trapping of holocytochrome c in the membrane. The problems of permeability of the outer mitochondrial membrane to apocytochrome c and the site and nature of the reaction by which the heme group is linked to the apoprotein are discussed.     

Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria.

1. A mutant of the iso-1-cytochrome c gene from Saccharomyces cerevisiae has been constructed which contains an Arg codon, replacing the normal trimethylated Lys at position 77. 2. This mutated gene was cloned into a pGem 1 vector and used for the in vitro translation of yeast iso-1-cytochrome c. 3. Utilizing an in vitro mitochondria binding assay, it was found that the mutant cytochrome c could transverse the yeast mitochondrial membrane, however the amount of protein incorporated was 3-fold less that of the trimethylated wild type. 4. Omission of the protein methyltransferase from assays containing the wild type cytochrome c caused only a slight reduction (15%) in the amount of protein incorporated. 5. These results suggest while the lysine residue 77 of apocytochrome c is important for mitochondria uptake, the methylation of this residue seems to play a relatively minor role.     

Role of cytochrome c heme lyase in the import of cytochrome c into mitochondria

The import of cytochrome c into Neurospora crassa mitochondria was examined at distinct stages in vitro. The precursor protein, apocytochrome c, binds to mitochondria with high affinity and specificity but is not transported completely across the outer membrane in the absence of conversion to holocytochrome c. The bound apocytochrome c is accessible to externally added proteases but at the same time penetrates far enough through the outer membrane to interact with cytochrome c heme lyase. Formation of a complex in which apocytochrome c and cytochrome c heme lyase participate represents the rate-limiting step of cytochrome c import. Conversion from the bound state to holocytochrome c, on the other hand, occurs 10-30-fold faster. Association of apocytochrome c with cytochrome c heme lyase also takes place after solubilizing mitochondria with detergent. We conclude that the bound apocytochrome c, spanning the outer membrane, forms a complex with cytochrome c heme lyase from which it can react further to be converted to holocytochrome c and be translocated completely into the intermembrane space.     

Apocytochrome c induces pH-dependent vesicle fusion

The ability of apocytochrome c and the heme containing respiratory chain component, cytochrome c, to induce fusion of phosphatidylcholine (PC) small unilamellar vesicles containing 0-50 mol % negatively charged lipids was examined. Both molecules mediated fusion of phosphatidylserine (PS):PC 1:1 vesicles as measured by energy transfer changes between fluorescent lipid probes in a concentration- and pH-dependent manner, although cytochrome c was less potent and interacted over a more limited pH range than the apocytochrome c. Maximal fusion occurred at pH 3, far below the pKa of the 19 lysine groups contained in the protein (pI = 10.5). A similar pH dependence was observed for vesicles containing 50 mol % cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylinositol (PI) in PC but the apparent pKa values varied somewhat. In the absence of vesicles, the secondary structure of apocytochrome c was unchanged over this pH range, but in the presence of negatively charged vesicles, the polypeptide underwent a marked conformational change from random coil to alpha-helix. By comparing the pH dependencies of fusion induced by poly-L-lysine and apocytochrome c, we concluded that the pH dependence derived from changes in the net charge on both the vesicles and apocytochrome c. Aggregation could occur under conditions where fusion was imperceptible. Fusion increased with increasing mole ratio of PS. Apocytochrome c did induce some fusion of vesicles composed only of PC with a maximum effect at pH 4. Biosynthesis of cytochrome c involves translocation of apocytochrome c from the cytosol across the outer mitochondrial membrane to the outer mitochondrial space where the heme group is attached. The ability of apocytochrome c to induce fusion of both PS-containing and PC-only vesicles may reflect characteristics of protein/membrane interaction that pertain to its biological translocation.     

Import of cytochrome c into mitochondria. Cytochrome c heme lyase

The import of cytochrome c into mitochondria can be resolved into a number of discrete steps. Here we report on the covalent attachment of heme to apocytochrome c by the enzyme cytochrome c heme lyase in mitochondria from Neurospora crassa. A new method was developed to measure directly the linkage of heme to apocytochrome c. This method is independent of conformational changes in the protein accompanying heme attachment. Tryptic peptides of [35S]cysteine-labelled apocytochrome c, and of enzymatically formed holocytochrome c, were resolved by reverse-phase HPLC. The cysteine-containing peptide to which heme was attached eluted later than the corresponding peptide from apocytochrome c and could be quantified by counting 35S radioactivity as a measure of holocytochrome c formation. Using this procedure, the covalent attachment of heme to apocytochrome c, which is dependent on the enzyme cytochrome c heme lyase, could be measured. Activity required heme (as hemin) and could be reversibly inhibited by the analogue deuterohemin. Holocytochrome c formation was stimulated 5--10-fold by NADH greater than NADPH greater than glutathione and was independent of a potential across the inner mitochondrial membrane. NADH was not required for the binding of apocytochrome c to mitochondria and was not involved in the reduction of the cysteine thiols prior to heme attachment. Holocytochrome c formation was also dependent on a cytosolic factor that was necessary for the heme attaching step of cytochrome c import. The factor was a heat-stable, protease-insensitive, low-molecular-mass component of unknown function. Cytochrome c heme lyase appeared to be a soluble protein located in the mitochondrial intermembrane space and was distinct from the previously identified apocytochrome c binding protein having a similar location. A model is presented in which the covalent attachment of heme by cytochrome c heme lyase also plays an essential role in the import pathway of cytochrome c.     

Trimethyllysine and protein function. Effect of methylation and mutagenesis of lysine 115 of calmodulin on NAD kinase activation

Unmethylated calmodulins have been enzymatically methylated at lysine 115, and a direct effect of this methylation on NAD kinase activation has been shown. Similar to naturally occurring calmodulins with trimethyllysine 115, the enzymatically methylated calmodulins activated an NAD kinase preparation to a maximal level that was at least 3-fold lower than the level of activation obtained with the corresponding unmethylated calmodulins. Methylation did not alter the cyclic nucleotide phosphodiesterase activator properties of these calmodulins. A genetically engineered calmodulin containing an arginine at position 115 instead of a lysine was produced by site-specific mutagenesis of a cloned synthetic calmodulin gene. The arginine derivative retained the higher maximal NAD kinase activator properties of the unmethylated calmodulins but was no longer susceptible to the effects of the methyltransferase. The data indicate that the reduction in the level of NAD kinase activation is the direct result of trimethylation of lysine 115 of calmodulin, provide a precedent for a functional effect of trimethyllysine in a protein, and raise the possibility that some of calmodulin's physiological activities may be affected by lysine methylation.     

CYC2 encodes a factor involved in mitochondrial import of yeast cytochrome c.

The gene CYC2 from the yeast Saccharomyces cerevisiae was previously shown to affect levels of mitochondrial cytochrome c by acting at a posttranslational step in cytochrome c biosynthesis. We report here the cloning and identification of the CYC2 gene product as a protein involved in import of cytochrome c into mitochondria. CYC2 encodes a 168-amino-acid open reading frame with at least two potential transmembrane segments. Antibodies against a synthetic peptide corresponding to the carboxyl terminus of the predicted sequence were raised. These antibodies recognize multiple bands on immunoblots of mitochondrial extracts. The intensities of these bands vary according to the gene dosage of CYC2 in various isogenic strains. Immunoblotting of subcellular fractions suggests that the CYC2 gene product is a mitochondrial protein. Deletion of CYC2 leads to accumulation of apocytochrome c in the cytoplasm. However, strains with deletions of this gene still import low levels of cytochrome c into mitochondria. The effects of cyc2 mutations are more pronounced in rho- strains than in rho+ strains, even though rho- strains that are CYC2+ contain normal levels of holocytochrome c. cyc2 mutations affect levels of iso-1-cytochrome c more than they do levels of iso-2-cytochrome c, apparently because of the greater susceptibility of apo-iso-1-cytochrome c to degradation in the cytoplasm. We propose that CYC2 encodes a factor that increases the efficiency of cytochrome c import into mitochondria.     

Enhanced crystal packing due to solvent reorganization through reductive methylation of lysine residues in oxidoreductase from <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Protein crystallization is in part driven by the changes in the entropy of the system, but opinions differ as to whether the solute (protein) or solvent (water) molecules make more of a contribution to the overall entropic change. Methylation of lysine residues in proteins has been used to enhance protein crystallization. We investigated using molecular dynamics simulations with explicit solvent molecules, the behavior of several native proteins and their methylated counterparts chosen from an earlier large-scale study. Methylated lysines are capable of making a variety of interactions including H-bonds with protein residues and solvent. We demonstrate that methylation on the lysine slightly increases its side chain conformational entropy by about 3.5 J mol⁻¹ K⁻¹. Analysis of the radial and spatial distributions of the water molecules around the methylated lysine surface in oxidoreductase from Streptococcus pneumoniae revealed a larger sphere of water molecules with low entropy, as compared with solvent associated with unmethylated lysine. If methylated lysine were to make interactions at the protein–protein interface, the low-entropy water molecules associated with methylated lysines would be released, resulting in a gain of entropy. We show that this gain more than compensates for the loss of protein entropy. Therefore, we propose that lysine methylation favors the formation of crystals through solvent entropic gain.     

Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase

The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024cΔ strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6).     

Iso-cytochrome c species from baker''s yeast. Analysis of their circular-dichroism spectra.

The circular-dichroism spectra of baker's-yeast iso-1- (methylated and unmethylated forms) and iso-2-cytochrome c species were examined between 200 and 600nm. In the visible region the yeast haemoproteins have characteristics nearly indistinguishable from those of horse heart cytochrome c. From the spectra in the u.v. region the latter appears, however, to be more helical. It is proposed that the likely element of non-helical structure in iso-1-cytochrome c is residues 62-70.     

Helix formation in methylated copolymers of lysine and alanine.

Random copolymers of lysine and alanine, 2:1 and 1:1, were trimethylated on the lysine amino groups to quaternary ammonium groups. Methylated and unmethylated polymers were prepared with Cl- or ClO4- as the counterion. CD spectra were measured for increasing concentration of peptide without added salt, and at constant peptide concentration in increasing NaCl or NaClO4. Unmethylated peptides, as the chloride, form alpha-helix more readily than do the methylated peptides. The opposite occurs with ClO4- as counterion. The helix-promoting effect of methylated lysine residues (ClO4- counterion) is diminished by the presence of alanine, as compared with effects when lysine is the only type of residue. The effect of methylation of proteins on helix formation may depend on the types of anionic groups with which the protein may be involved.     

Adding a Lysine Mimic in the Design of Potent Inhibitors of Histone Lysine Methyltransferases

Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the histone code. One common feature of these activities is the recognition of lysines in methylated and unmethylated states, whether they are substrates, reaction products, or binding partners. We applied the concept of adding a lysine mimic to an established inhibitor (BIX-01294) of histone H3 lysine 9 methyltransferases G9a and G9a-like protein by including a 5-aminopentyloxy moiety, which is inserted into the target lysine-binding channel and becomes methylated by G9a-like protein, albeit slowly. The compound enhances its potency in vitro and reduces cell toxicity in vivo. We suggest that adding a lysine or methyl-lysine mimic should be considered in the design of small-molecule inhibitors for other methyl-lysine writers, erasers, and readers.     

Role of lysine methylation in the activities of elongation factor 1 alpha

Previous work in our laboratory has demonstrated that 19% of the lysine residues in the protein synthesis elongation factor (EF-1 alpha) are methylated when the factor is purified from the mycelial form of the fungus Mucor racemosus. However, the same factor, when purified from spores of M. racemosus, is largely unmethylated. Despite its wide-spread occurrence in a great number of basic proteins, the functional significance of lysine N-methylation remains poorly understood. Spore and mycelial forms of EF-1 alpha were therefore compared in a series of assays to determine their relative affinities for various substrates and cofactors known to interact with the factor during the elongation cycle. The results suggested that hypomethylated and fully methylated EF-1 alpha had equal affinities for GTP, aminoacyl-tRNA, and ribosomes. Also, methylation did not appear to affect the accuracy of translation in an in vitro system. However, experiments did suggest that methylation may affect the ability of the factor to form complexes with other subunits (EF-1 beta gamma) which are known to enhance the overall rate of protein synthesis.     

Insertion of apocytochrome c into lipid vesicles

Apocytochrome c (cytochrome c without the heme) is synthesized in the cell cytoplasm without a cleaved signal sequence, then transported across the outer mitochondrial membrane. We have studied the interaction of apocytochrome c with lipid vesicles as a model for understanding protein translocation across membranes. Apocytochrome c (but not holocytochrome c) that has been incubated with vesicles at 37 degrees C in 0.2 M NaCl binds to the vesicles. Under these conditions, as well as upon incubation with detergent or at high protein concentrations, all the added protein remains partly accessible to externally added protease, but a COOH-terminal fragment of some of the protein molecules becomes protected against digestion. When apocytochrome c is added to azolectin vesicles with internally trapped proteases, most of the added protein can be digested, even in the presence of a large excess of protease inhibitor external to the vesicles. Thus, in spite of a lack of nonpolar stretches in its amino acid sequence, apocytochrome c is capable of binding to and inserting into lipid membranes. In this model system, transport may be driven by trapping of protease-digested apocytochrome c on one side of the membrane.