Protein synthesis in cultured muscle cells: methylation of nascent proteins

Protein methylation was examined in primary cultures of rat leg muscle cells between 7 and 9 days of culture. Methyl[¹⁴C]- or [³H]-methionine was introduced into the culture medium and the cells were sampled for radioactive methylated protein residues. Incorporation of the total radioactivity was linear for at least 4 hr after introduction of the methionine label. When labeling was studied for periods between 10–30 min, the methylation of polyribosome-bound, presumably nascent, proteins was unaffected by addition of cycloheximide to the culture medium. The antibiotic, however, inhibited incorporation of methionine, and consequently increased the ratios of the incorporated methylated, to methionine residues and the ratio of ribosome-bound to free radioactivity. The methylated, polyribosome-bound proteins were decreased when puromycin was added to the culture medium. It is proposed that selective methylation of nascent proteins, such as myosin, can begin at the level of polyribosomes and be completed in the cytosol of muscle cells cultured in vitro.     

Effect of methylation on the stability of cytochrome c of Saccharomyces cerevisiae in vivo

The in vivo stability of methylated and unmethylated cytochrome c in Saccharomyces cerevisiae was studied by pulse-labeling the hemoproteins with [methyl-3H]-methionine and/or [2-14C]methionine and following the fate of these proteins under anaerobiosis and in the presence of cycloleucine. These two conditions will respectively block further cytochrome c synthesis and inhibit methylation by lowering the cellular S-adenosyl-L-methionine pool and, thus, permit an unambiguous interpretation of the data. The results showed that the rate of degradation of unmethylated cytochrome c was constant throughout the chase period, while methylated cytochrome c degradation was seen only in the later part of cold chase. At the end of the chase period (40 h), the extent of degradation of the unmethylated species was three times higher than the methylated species. This indicated that the methylation of cytochrome c has a protective effects against the intracellular proteolytic enzyme attack on itself. Furthermore, this protective effect was considerably reduced in the petite mutant, which lacks high affinity cytochrome c binding sites, functional cytochrome c reductase, and oxidase, and possesses a less integrated and organized mitochondrial membrane. These results led us to the conclusion that the mechanism of methylated cytochrome c stabilization is best explained by a higher efficacy of binding to the mitochondria.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Biosynthesis of 3-dimethylsulfoniopropionate in Wollastonia biflora (L.) DC. Evidence that S-methylmethionine is an intermediate.

The compatible solute 3-dimethylsulfoniopropionate (DMSP) is accumulated by certain salt-tolerant flowering plants and marine algae. It is the major biogenic precursor of dimethylsulfide, an important sulfur-containing trace gas in the atmosphere. DMSP biosynthesis was investigated in Wollastonia biflora (L.) DC. [= Wedelia biflora (L.) DC., Melanthera biflora (L.) Wild, Asteraceae]. After characterizing DMSP and glycine betaine accumulation in three diverse genotypes, a glycine betaine-free genotype was chosen for radiotracer and stable isotope-labeling studies. In discs from young leaves, label from [U-14C]methionine was readily incorporated into the dimethylsulfide and acrylate moieties of DMSP. This establishes that DMSP is derived from methionine by deamination, decarboxylation, oxidation, and methylation steps, without indicating their order. Five lines of evidence indicated that methylation is the first step in the sequence, not the last. (a) In pulse-chase experiments with [14C]methionine, S-methylmethionine (SMM) had the labeling pattern expected of a pathway intermediate, whereas 3-methylthiopropionate (MTP) did not. (b) [14C]SMM was efficiently converted to DMSP but [14C]MTP was not. (c) The addition of unlabeled SMM, but not of MTP, reduced the synthesis of [14C]DMSP from [14C]methionine. (d) The dimethylsulfide group of [13CH3,C2H3]SMM was incorporated as a unit into DMSP. (e) When [C2H3,C2H3]SMM was given together with [13CH3]methionine, the main product was [C2H3,C2H3]DMSP, not [13CH3,C2H3]DMSP or [13CH3,13CH3]DMSP. The stable isotope labeling results also show that the SMM cycle does not operate at a high level in W. biflora leaves.     

Methylation of ribosomal proteins in Bacillus subtilis

We measured the methylation of ribosomal proteins from the 30S and 50S subunits of Bacillus subtilis after growing the cells in the presence of [1-14C]methionine and [methyl-3H]methionine. Two-dimensional polyacrylamide gel electrophoretic analysis revealed a preferential methylation of the 50S ribosomal proteins. Proteins L11 and L16, and possibly L9, L10, L18, and L20, were methylated. On the other hand, only two possibly methylated proteins were found on the 30S subunit. A comparison of these results with those for Escherichia coli suggests a common methylation pattern for the bacterial ribosomal proteins.     

Methylation of elongation factor 1α in mouse 3T3B and 3T3BSV40 cells

Two-dimensional gel electrophoretic (NEPHGE) analysis of proteins from mouse 3T3B and 3T3B/SV40 cells labelled with [methyl-³H]methionine in the presence of cycloheximide have revealed that the elongation factor 1α (EF-1α) in these cells is methylated and that the extent of methylation is higher in the SV40 transformed cell type. It is suggested that methylation may account for differences in growth properties for the different cell types.     

Metabolism of methionine and biosynthesis of caffeine in the tea plant (Camellia sinensis L.).

1. Caffeine biosynthesis was studied by following the incorporation of 14C into the products of L-[Me-14C]methionine metabolism in tea shoot tips. 2. After administration of a 'pulse' of L-[Me-14C]methionine, almost all of the L-[Me-14C]methionine supplied disappeared within 1 h, and 14C-labelled caffeine synthesis increased throughout the experimental periods, whereas the radioactivities of an unknown compound and theobromine were highest at 3 h after the uptake of L-[Me-14C]methionine, followed by a steady decrease. There was also slight incorporation of the label into 7-methylxanthine, serine, glutamate and aspartate, disappearing by 36 h after the absorption of L-[Me-14C]methionine. 3. The radioactivities of nucleic acids derived from L-[Me-14C]methionine increased rapidly during the first 12 h incubation period and then decreased steadily. Sedimentation analysis of nucleic acids by sucrose-gradient centrifugation showed that methylation of nucleic acids in tea shoot tips occurred mainly in the tRNA fraction. The main product among the methylated bases in tea shoot tips was identified as 1-methyladenine. 4. The results indicated that the purine ring in caffeine is derived from the purine nucleotides in the nucleotide pool rather than in nucleic acids. A metabolic scheme to show the production of caffeine and related methylxanthines from the nucleotides in tea plants is discussed.     

Carotenoid biosynthesis in Rhodopseudomonas spheroides. S-Adenosylmethionine as the methylating agent in the biosynthesis of spheroidene and spheroidenone

[methyl-(14)C]Methionine and S-adenosyl[methyl-(14)C]methionine were incorporated into the methoxycarotenoids spheroidene and spheroidenone by Rhodopseudomonas spheroides. The incorporation was greatly enhanced in the presence of lysozyme. On degradation of labelled spheroidene by hydriodic acid, the (14)C label was recovered in methyl iodide. Degradation of spheroidenone by reduction and allylic dehydration and demethylation of the reduction product gave a mixture of unlabelled carotenoid hydrocarbons, including 3,4-didehydrolycopene and 3,4-didehydro-7',8'-dihydrolycopene. The label from [methyl-(14)C]methionine and S-adenosyl[methyl-(14)C]methionine was located specifically in the methoxy group of spheroidene and spheroidenone. The biosynthesis of methoxycarotenoids in Rps. spheroides involves methylation of the tertiary hydroxyl groups of intermediates with S-adenosylmethionine.     

Modification of yeast ribosomal proteins. Methylation.

Two-dimensional polyacrylamide-gel electrophoretic analysis of yeast ribosomal proteins uniformly labelled in vivo with [methyl-3H]methionine and [1-14C]methionine revealed that four ribosomal proteins are methylated, i.e. proteins S31, S32, L15 and L41. Lysine and arginine appear to be the predominant acceptors of the methyl groups. The degree of methylation ranges from 0.09 to 0.20 methyl group per modified ribosomal protein species.     

Secretory Proteins from Adrenal Medullary Cells Are Carboxyl-Methylated In Vivo and Released Under Their Methylated Form by Acetylcholine

The carboxyl methylation of secretory proteins in vivo was investigated in bovine adrenal medullary cells in culture. Chromogranin A, the major intragranular secretory protein in adrenal medullary cells, and other secretory proteins were found to be carboxyl-methylated within secretory vesicles. The in vivo labeling pattern using [methyl-3H]methionine and the in vitro labeling pattern using S-adenosyl-[methyl-14C]methionine of intravesicular secretory proteins were similar. The detection of methylated chromogranin A in mature secretory vesicles required 3-6 h, a time consistent with the synthesis and storage of secretory proteins in this tissue. Carboxyl-methylated chromogranin A was secreted from medullary cells by exocytosis via activation of nicotinic cholinergic receptor and recovered still under the methylated form in the incubation medium. Since protein-carboxyl-methylase is cytosolic, these results suggest that methylation of secretory proteins is a cotranslational phenomenon.     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

Cytoplasmic location of undermethylated messenger RNA in Novikoff cells.

Novikoff cells in culture were labeled with L-[methyl-3H]methionine and [U-14C]uridine in the presence of (a) TubHcy2, (b) AdoHcy, (c) homocysteine, (d) tubercidin, or (e) without any additions. Only in cultures labeled in the presence of TubHcy were undermethylated cap structures observed to represent a significant portion of [3H]methyl radioactivity. Novikoff cells in culture were then simultaneously labeled with L-[methyl-3H]methionine and [32P]orthophosphate in the presence or absence of TubHcy. Total cytoplasmic, polysomal and monosomal poly(A)-containing RNAs were analyzed. Both monosomal and polysomal mRNA fractions from TubHcy-treated cells contain partially methylated cap structures, suggesting that 2'-O-methylation of the nucleoside adjacent to the pyrophosphate linkage in caps is not required for transport, ribosomal binding or translation. Comparison of nuclear and cytoplasmic cap structures from normal and inhibited cultures indicate that an altered mRNA population is generated in the presence of TubHcy.     

Biosynthesis of carnitine and 4-N-trimethylaminobutyrate from lysine

The conversion of l-[U-(14)C]lysine into carnitine was demonstrated in normal, choline-deficient and lysine-deficient rats. In other experiments in vivo radioactivity from l-[4,5-(3)H]lysine and dl-[6-(14)C]lysine was incorporated into carnitine; however, radioactivity from dl-[1-(14)C]lysine and dl-[2-(14)C]lysine was not incorporated. Administered l-[Me-(14)C]methionine labelled only the 4-N-methyl groups whereas lysine did not label these groups. Therefore lysine must be incorporated into the main carbon chain of carnitine. The methylation of lysine by a methionine source to form 6-N-trimethyl-lysine is postulated as an intermediate step in the biosynthesis of carnitine. Radioactive 4-N-trimethylaminobutyrate (butyrobetaine) was recovered from the urine of lysine-deficient rats injected with [U-(14)C]lysine. This lysine-derived label was incorporated only into the butyrate carbon chain. The specific radioactivity of the trimethylaminobutyrate was 12 times that of carnitine isolated from the urine or carcasses of the same animals. These data further support the idea that the last step in the formation of carnitine from lysine was the hydroxylation of trimethylaminobutyric acid, and are consistent with the following sequence: lysine+methionine --> 6-N-trimethyl-lysine --> --> 4-N-trimethylaminobutyrate --> carnitine.     

Continuous equilibration of phosphatidylcholine and its precursors between endoplasmic reticulum and mitochondria in yeast

In Saccharomyces cerevisiae phosphatidylcholine (PC) is synthesized in the ER and transported to mitochondria via an unknown mechanism. The transport of PC synthesized by the triple methylation of phosphatidylethanolamine was investigated by pulsing yeast spheroplasts with l-[methyl-3H]methionine, followed by a chase with unlabeled methionine and subcellular fractionation. During the pulse, increasing amounts of PC and its mono- and dimethylated precursors (PMME and PDME, respectively) appear in similar proportions in both microsomes and mitochondria, with the extent of incorporation in microsomes being twice that in mitochondria. During the chase, the [3H]-methyl label from the precursors accumulates into PC with similar kinetics in both organelles. The results demonstrate that transport of methylated phospholipids from ER to mitochondria is 1) coupled to synthesis, 2) not selective for PC, 3) at least as fast as the fastest step in the methylation of PE, and 4) bidirectional for PMME and PDME. The interorganellar equilibration of methylated phospholipids was reconstituted in vitro and did not depend on ongoing methylation, cytosolic factors, ATP, and energization of the mitochondria, although energization could accelerate the reaction. The exchange of methylated phospholipids was reduced after pretreating both microsomes and mitochondria with trypsin, indicating the involvement of membrane proteins from both organelles.     

Effects of estradiol on uterine ribonucleic acid metabolism. Assessment of transfer ribonucleic acid methylation.

Immature rats treated with estradiol for selected periods of time demonstrated both increased methylation of uterine transfer ribonucleic acid (tRNA) and methylase activities. Whereas the former parameter was assessed by incubating whole uteri with [methyl-14C]methionine and measuring the incorporation of isotope into the tRNA, methylase activity was obtained by measuring the rate of incorporation of methyl groups from S-adenosyl[methyl-14C]methionine into heterologous tRNA (Escherichia coli B) in the presence of uterine cytosol preparations (100,000g supernatants). Although increased methylation of tRNA during the estrogen response was demonstrated, additional studies indicated that these results were largely attributable to an increased rate of synthesis of tRNA rather than gross changes in either the type or amount of methylated constituents present. Evidence in this regard included the inability of estrogen treatment of alter significantly the (a) resulting patterns of methyl-14C-methylated constituents of uterine tRNA, (b) the extent ot which [2-14C]guanine residues, incorporated into tRNA, become methylated, (c) the extent of methylation of precursor tRNA in the absence of tRNA synthesis, and (d) the types of methylase activities expressed in vitro.     

Isoprenylation and carboxylmethylation in small GTP-binding proteins of pheochromocytoma (PC-12) cells

1. A group of 21 to 24-kDa proteins of pheochromocytoma (PC-12) cells was found in blot overlay assays to bind specifically [alpha-32P]GTP. Binding was inhibited by GTP analogues but not by ATP. Such small GTP-binding proteins were found in the cytosolic and in the particulate fraction of the cells, but they were unevenly distributed: about 75% of the small GTP-binding proteins were localized within the particulate fraction of the cells. Separation of these proteins by two-dimensional gel electrophoresis revealed the existence of seven distinct [alpha-32P]GTP-binding proteins. 2. Targeting of the small GTP-binding proteins to the particulate fraction of PC-12 cells requires modification by isoprenoids, since depleting the cells of the isoprenoid precursor mevalonic acid (MVA) by the use of lovastatin resulted in a 50% decrease in membrane-bound small GTP-binding proteins, with a proportionate increase in the cytosolic form. This blocking effect of lovastatin was reversed by exogenously added MVA. 3. In addition, metabolic labeling of PC-12 cells with [3H]MVA revealed incorporation of [3H]MVA metabolites into the cluster of 21 to 24-kDa proteins in a form typical of isoprenoids; the label was not removed from the proteins by hydroxylamine, and labeling was enhanced in cells incubated with lovastatin. The latter effect reflects a decrease in the isotopic dilution of the exogenously added [3H]MVA, as the addition of exogenous MVA reversed the effect of lovastatin on [3H]MVA-metabolite incorporation into the 21 to 24-kDa proteins. 4. Additional experiments demonstrated that isoprenylation is required not only for membrane association of small GTP-binding proteins, but also for their further modification by a methylation enzyme. This was evident in experiments in which the cells were metabolically labeled with [methyl-3H]methionine, a methylation precursor. The group of 21 to 24-kDa proteins was labeled with a methyl-3H group in a form typical of C-terminal-cysteinyl carboxylmethyl esters. Their methylation was blocked by the methylation inhibitors methylthioadenosine (MTA), 3-deazadenosine and homocysteine thiolactone as well as by lovastatin. MVA reversed the lovastatin block of methylation. 5. Two-dimensional gel analysis of the [3H]methylated proteins detected seven methylated small GTP-binding proteins that correspond to the isoprenylated proteins. Levels of the small GTP-binding proteins as well as isoprenylation and methylation were reduced by cycloheximide. 6. Distribution of the methylated proteins between particulate and cytosolic fractions was found to be similar to that of the small GTP-binding proteins (i.e., a 4:1 ratio).(ABSTRACT TRUNCATED AT 400 WORDS)     

Methylation of phospholipids in microsomes of the rat aorta

The methylation of phospholipids by S-adenosyl-L-methionine was characterized in microsomes prepared from strips of rat aorta. In the presence of 0.5 microM S-adenosyl-L-methionine, endogenous phosphatidylethanolamine was methylated to form three products: phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. In the presence of 150 microM S-adenosyl-L-methionine the methylation activity increased more than 50-fold and the principal radioactive product was phosphatidylcholine. Optimal activity was at pH 9 and no magnesium requirement was detected. Exogenous phosphatidylethanolamine, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine served as substrates for the enzyme. The methylation of exogenous phosphatidyl-N,N-dimethylethanolamine proceeded at a slower rate. Incubation of trypsin with the aorta microsomes reduced the enzymatic activity and reduced the relative yield of phosphatidyl-N-monomethylethanolamine. Phospholipase C degraded the methylated phospholipids, but phosphatidyl-N,N-dimethylethanolamine appeared to be less accessible to the phospholipase. The phospholipid methylation activity was inhibited by the addition of S-adenosyl-L-homocysteine or by L-homocysteinethiolactone. When intact strips of rat aorta were incubated with L-[methyl-3H]methionine, [3H]methyl groups were incorporated into phospholipids. This incorporation was inhibited when L-homocysteinethiolactone was added to the incubation. Polarized fluorescence of diphenylhexatriene in aorta microsomes was measured to determine the apparent membrane fluidity. When intact strips of aorta were incubated with methionine or with L-homocysteinethiolactone, methionine enhanced and L-homocysteinethiolactone decreased apparent fluidity of the microsomal membranes. Phospholipid methylation activity was examined in aorta microsomes prepared from genetically spontaneous hypertensive SHR strain rats. Phospholipid methylation activity was substantially greater in the SHR aorta microsomes than in microsomes prepared from Wistar-Kyoto WKY control strain aorta. Membrane fluidity was greater in the SHR aorta microsomes than in the WKY aorta microsomes. The hypothesis that phospholipid methylation activity influences fluidity of membranes and the possible involvement of methylated phospholipids in aorta membrane functions are discussed.