WHAT IS THE FUNCTION OF PROTEIN CARBOXYL METHYLATION?
AREN VAN WAARDE*
Cell Biology and Morphogenesis Unit, Laboratory of Zoology, University of Leiden, Kaiserstraat 63, 2311 GP Leiden, The Netherlands
(Received 14 April 1986)
Abstract--The following functions of protein carboxyl methylation seem to be reasonably well established: 1. Multiple, stoichiometric methylation of chemotactic receptors in bacteria at glutamyl residues serves
as one (but not the only) adaptation mechanism of the transduction chain to constant background levels of chemotactic stimuli.
2. Stoichiometric methylation of hormones and hormone carrier proteins plays a role in hormone storage and secretion by the pituitary gland.
3. Substoichiometric methylation at o-aspartyl residues is involved in a repair mechanism of aged proteins.
4. Stoichiometric methylation of calmodulin modulates the sensitivity of calmodulin-dependent pro- cesses to calcium.
Research of the past 3 years has indicated that in order to demonstrate an involvement of methylation in the coupling of surface receptors to intracellular events three new criteria have to be met: (a) the cell should possess a protein carboxyl methylase with relatively narrow substrate specificity; (b) methylation should take place at L-amino acid residues; (c) the methyl accepting proteins should be methylated in a stoichiometric fashion.
I. INTRODUCTION
Carboxyl methylation is a biochemical reaction in which free carboxyl groups of proteins are trans- formed into O-methyl esters. Methylation is a revers- ible pathway for post-translational protein modification, resembling the better known process of phosphorylation (Kim, 1977; Paik and Kim, 1980).
The mechanism of carboxyl methylation is presented in Fig. 1. Proteins are esterified by the action of a specific enzyme, protein carboxyl methyl- ase (PCM), which uses S-adenosylmethionine (Ado- Met) as a methyl donor. Protein methyl esters can subsequently be hydrolyzed by the action of a second enzyme, protein methyl esterase (PME), which re- leases the original protein and methanol as end- products.
Methanol has for a long time been known to be a minor consituent of the blood and the expired air of human subjects, whereas a so-called "methanol- forming enzyme" has been found in many vertebrate tissues during the sixties (Axelrod and Cohn, 1971). The identification of the "methanol-forming enzyme" as a protein methyl transferase, however, occurred in 1969-1970, when the enzyme was purified from calf spleen and bovine thymus (Liss et al., 1969; Kim and
*Present address: Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 6666, New Haven, CT 06511, USA.
Paik, 1970). Several proteins (ribonuclease, oval- bumin, gelatin, gamma-globulin and histone IIa) could act as substrates. The resulting methyl esters were found to be very labile at neutral and alkaline pH (Kim and Paik, 1970). The hydrolysis product
was identified as methanol by chemical modification with 3,5-dinitrobenzoylchloride and by co-elution with authentic methanol during gas chromatography (Liss et al., 1969).
Since 1970, several purification procedures have been described for PCM (Aswad and Deight, 1983a; Diliberto and Axelrod, 1974; Kim et al., 1983; Paik and Kim, 1980; Trivedi et al., 1982; Ullah and Ordal, 1981). A complex scheme of purification is sometimes necessary to obtain an enzyme preparation which is completely free from endogenous substrate (Kim and Paik, 1970; Ullah and Ordal, 1981). The purified enzyme shows a high affinity for AdoMet (1-5/zM) and a high specificity for AdoMet as a methyl donor (Ciaranello et al., 1972; Diliberto and Axelrod, 1974; Kim and Paik, 1970; Trivedi et al., 1982; Ullah and Ordai, 1981). It is extremely sensitive to product inhibition by S-adenosylhomocysteine (AdoHcy, see Fig. I), apparent inhibition constants being in the range of 0.2-1.5 #M (Trivedi et al., 1982; Ullah and Ordal, 1981). The activity has been demonstrated in all vertebrate tissues examined, the greatest amount of enzyme being present in brain, testes and hypo- thalamus (Axelrod and Cohn, 1971; Ciaranello et al., 1972; Diliberto and Axelrod, 1976; Paik et al., 1971). PCM is also present in bacteria (Springer et al., 1979; Hazelbauer, 1979; Springer and Koshland, 1977). In rat adrenal gland, only a single PCM species is observed in iso-electric focusing (Cusan et al., 1981a), but testis and brain contain a number of iso-enzymes with different iso-electric points (Aswad and Deight, 1983a; Cusan et al., 1981a).
The nature of the methylated amino acid residues seems to be different in different tissues. Calf brain PCM has been reported to methylate corticotropin at a specific glutamyl residue. Kim and Li (1979b) labeled ACTH with ~aC-AdoMet and subjected the hormone subsequently to pepsin digestion. The digest was analyzed by paper electrophoresis, which showed one specific peptide (the alpha-S ACTH (6-28)) to be labeled. Since this peptide contains only one free carboxyl group at Glu-28, this residue should be the one modified by methylation. ACTH-methylation appeared to be stoichiometrical, levels of 30 mol% of esterification being reached in vitro (Kim and Li, 1979b).
The methylated amino acid residues in bacterial membranes have also been identified as stoichiomet- rically formed glutamic acid gamma-methyl esters (Ahlgren and Ordal, 1983; Kleene et al., 1977; Stock and Koshland, 1981).
In contrast with these observations on prokaryotes and pituitary gland, the methylated residues of eryth- rocyte membranes have been proved to be aspartic acid beta-methyl esters. Janson and Clarke (1980) subjected the membranes after labeling with radio- active S-adenosylmethionine to digestion by papain and carboxypeptidase Y. Subsequently, the radio- labeled products were separated by ion-exchange chromatography. Labeled peaks occurred at the pos- itions of methanol, aspartic acid beta-methyl ester and unhydrolyzed protein, but no activity co-eluted with authentic glutamic acid gamma-methyl ester. More recently, the aspartyl residues of erythrocyte membranes have been shown to be in the unusual D-configuration. Clarke and co-workers subjected the
proteolyticaUy derived aspartic acid beta-methyl ester to treatment with L- and D-amino acid oxidases. They found it to be broken down by the D-enzyme, but not by L-amino acid oxidase. L-Leucyl-aspartic acid beta- methyl ester was synthesized from erythrocyte digests and the product was shown to elute at the position of the D-aspartic acid stereoisomer in ion exchange chromatography. Methylation of erythrocyte mem- brane proteins was always very substoichiometric (levels less than 0.02 mol% of esterification being reached during in vitro methylation of erythrocyte ghosts), which indicates only a very low percentage of the aspartyl residues to be in the D-configuration (McFadden and Clarke, 1982; McFadden et al., 1983; O'Connor and Clarke, 1982, 1983).
Bacteria have been shown to contain a chemotactic PCM with very narrow substrate specificity, a small class of 60,000 dalton inner membrane proteins being the only effective methyl acceptors (Clarke et al., 1980; Springer and Koshland, 1977). The specificity of this bacterial enzyme is so high, that a purified preparation of Bacillus subtilis is unable to methylate Escherichia coli membranes (Ullah and Ordal, 1981). Besides the specific chemotactic enzyme, however, E. coli and Salmonella typhimurium also contain a PCM with low substrate specificity (Clarke et al., 1980; Kim et al., 1977). The vertebrate enzymes which have been characterized thus far can methylate a large number of different substrates (Diliberto and Axel- rod, 1974; Kim and Paik, 1970; Liss et al., 1969; Clarke and O'Connor, 1983).
PCM-activity in Bacillus subtilis seems to be regu- lated by the level of free calcium, which is a com- petitive inhibitor of the purified enzyme with an apparent inhibition constant of 65 nM (Ullah and Ordal, 1981). In Halobacterium halobium, methyl- ation is also inhibited by free calcium (Schimz, 1982). Similar evidence has not yet been presented for any eukaryote PCM, however, whereas the enzyme from calf thymus is not affected by calcium ion up to a concentration of 2 mM (Paik and Kim, 1980).
In all vertebrate tissues studied, PCM has been shown to be a cytosolic enzyme (Bouchard et al., 1980; Chene et al., 1982; Diliberto and Axelrod, 1976; Edgar and Hope, 1976; Gagnon and Axelrod, 1979; O'Dea et al., 1978a,b,c). A small activity has been reported to exist in isolated nuclei of liver, brain and thymus (Quick et al., 1981) and in brain syn- aptosomes (Diliberto and Axelrod, 1976). Prokaryote PCM is also localized in the soluble fraction of the cell (Panasenko and Koshland, 1979).
Hydrolysis of protein methyl esters was originally thought to be spontaneous, but Stock and Koshland (1978) were first to demonstrate the presence of a protein methyl esterase in bacteria. The enzyme was later purified from Bacillus subtilis. It requires di- valent cations (Mg) for activity, an optimum being observed at a magnesium concentration of 1.1 mM. The protein has a high affinity for its methylated substrate (10nM, Goldman et al., 1984). In hom- ogenates of Escherichia coli, PME-activity has been shown to be regulated by cyclic GMP, which causes strong inhibition whereas other nucleotides are with- out effect (Black et al., 1980, 1982).
A protein methyl esterase has also been observed in several vertebrate tissues, the greatest amount of
What is the function of protein carboxyl methylation? 425
enzyme being found in kidney (Chene et al., 1982; Gagnon, 1979). The bacterial and leukocyte methyl- esterases are almost exclusively cytosolic (Goldman et al., 1984; Panasenko and Koshland, 1979; Venk- atasubramanian et al., 1980), but in most organs of vertebrates, the major part of enzyme activity is associated with the particulate fraction (Bouchard et aL, 1980; Chene et aL, 1982; Gagnon, 1979).
The substrates for PCM and PME seem in all cases to be localized both in the soluble and particulate fractions of the cell (Aswad and Deight, 1983b; Bouchard et al., 1980; Edgar and Hope, 1976; Gag- non and Axelrod, 1979; O'Connor and Clarke, 1984; O'Dea et al., 1978a; Ridgway et al., 1977).
If. CARBOXYL METHYLATION DURING CHEMOTAXIS
11.1. Prokaryotes
Concerning the biological function of methylation, most information has been obtained for bacteria, since these can be easily cultivated in large quantities, several mutants are readily available and the methyl esters of glutamate residues in bacterial proteins are relatively stable (Panasenko and Koshland, 1979). Especially the flagellar enteric bacterium Escherichia coli has been the subject of extensive research in many laboratories (Hazelbauer et al., 1982).
II.1.1. Escherichia coli. In 1975, Kort et al. re- ported that the methyl group of 14C-methyl methi- onine is rapidly incorporated into 62,000 dalton pro- teins, which are localized in the inner membrane of the cell. When E. coli is stimulated with a chemo- attractant, methylation is increased from a basal level to a new value, which is reached within 2 min. Several non-chemotactic mutants were found to be defective also in this methylation response, which suggests the existence of a relationship between methylation and chemotaxis.
E. coli cells are propagated by flagella, which can rotate either clockwise or counterclockwise. In the absence of any chemical gradient, the bacteria swim in straight lines, which are interrupted at different intervals of time by an instant of uncoordinated tumbling due to flagellar reversal. After tumbling, the cells resume swimming in a randomly chosen new direction. When an attractant is locally introduced into the cell suspension, the tumbling behaviour is significantly changed. If the cell swims towards a higher concentration of attractant, the frequency of tumbling is very much decreased, but this response does not occur when the bacterium is moving away from the attractant source. Due to this mechanism, the cell spends more time going in the proper direc- tion than it does in other directions, positive chemo- taxis being the result. Repellents have the opposite effect, a positive gradient of repellent being able to increase the tumbling frequency, so that negative chemotaxis occurs (Springer et al., 1979; Oosawa and Imae, 1984).
When a constant level of stimulus is applied by homogeneous distribution of a non-metabolizable attractant over the cell suspension, the bacteria show a temporal decrease of their tumbling frequency, but they resume their original amount of tumbling within
a relatively short period of time. A further increase of the attractant concentration gives again rise to a temporal suppression of tumbling. Because of this behavior, the cells adapt to constant background levels of chemotactic stimuli, whereas they remain sensitive to positive stimulus gradients. When the stimulus is removed, bacteria have been shown to deadapt rapidly. Reintroduction of the same amount of attractant does not induce any change of tumbling when the cells are still adapted to the first stimulus. After deadaptation, however, a second stimulus will induce the same suppression of tumbling as occurred after the first addition of attractant (Springer et al., 1979).
Silverman and Simon (1977a) have studied several deletion mutants in order to determine the nature of polypeptides in E. coli which are necessary for chemotaxis. They described eight different gene dele- tions which resulted in a chemotaxis deficient pheno- type: che A (product 77 kD and 66 kD polypeptides), che B (38kD polypeptide), che D (64kD poly- peptide), che M (63, 61 and 60 kD polypeptides), the W (12 kD polypeptide), ehe X (28 kD polypeptide), the Y (8 kD polypeptide) and che Z (24 kD poly- peptide) (see Table 1). The synthesis of all these gene products was found to be regulated in parallel with that of the structural elements of the flagella.
Three of the polypeptides were subsequently identified as methyl accepting chemotaxis proteins (MCP's) . M C P 1 is lacking in the D mutants, M C P 2 in the M mutants and M C P 3 in the che Z strain. The methylation of M C P 1 was shown to be stimu- lated by application of the attractant serine, that of M C P 2 by aspartate. M C P 1 and M C P 2 are localized in the inner membrane of the cell. Mutants lacking M C P I are defective in their taxis for serine (this phenotype being called tsr), whereas mutants lacking M C P 2 are defective in their taxis towards aspartate (phenotype tar; Ridgway et al., 1977; Silverman and Simon, 1977b). Springer et al. (1977b) further identified the nature of the chemotactic sig- nals which are processed via the proteins M C P I and M C P 2. They found methylation of M C P 1 to be increased by the attractants serine and alpha- aminoisobutyrate, whereas it was decreased upon addition of the repellents acetate, benzoate, indole and leucine. The methylation level of M C P 2 was increased by the attractants aspartate and maltose, but decreased by the repellents nickel and cobalt.
On the basis of these observations it was concluded that the methyl accepting proteins are necessary for sensory excitation, since their absence in deletion mutants results in a lack of response to certain chemoeffectors. The function of methylation of these proteins was subsequently studied by Springer et al. (1977a) and Goy et al. (1977, 1978). The authors used methionine auxotrophic mutants, which are unable to synthesize methionine from precursors and therefore require the presence of methionine in the medium. In methionine-starved cells of this type, they could show that methionine was required for adaptation of the cells to increases in the concentration of chemical attractants, but the amino acid was not necessary for the maintenance of the adapted state or for deadaptation after removal of the stimulus. In a parallel series of experiments, they demonstrated that
426 ARES VAN WAARDE
an increase in the level of MCP-methylation requires methionine, but the maintenance of a constant methylation level or the demethylation which occurs after the removal of an attractant did not require the presence of the amino acid. They concluded that the methyl acceptors are transducers for the chemo- sensory signals, whereas the methylation of these proteins causes adaptation of the transduction chain to constant background levels of chemical stimuli.
Another chemotactic mutant was characterized by Parkinson (1978), Parkinson and Revello (1978) and Goy et al. (1977). Che X mutants were found to respond to both attractants and repellents with a change in tumbling frequency, but they were unable to terminate these behavioral changes as long as the simulating chemical was present. Therefore, che X cells show a constant or very much prolonged depres- sion of tumbling in the presence of attractants and a constant stimulation of tumbling in the presence of repellents, not only of those whose signals are pro- cessed via M C P I, but also of those of which the sensing requires the presence of M C P 2. Parkinson and Revelio (1978) suggested that the che X product could be a protein carboxyl methylase and this suggestion was confirmed by Goy et al. (1977), who demonstrated PCM-activity in the che X strain to be less than 5% of that observed in the wild type.
The mechanism of chemoeffector-induced changes in the level of MCP-methylation has been further elucidated by Kleene et al. (1979). Membrane vesicles were produced by treatment of bacteria with a French pressure cell and after dialysis for the removal of endogenous effectors and substrates, the prepara- tion was labeled by incubation with 14C-S-adenosyl- methionine. Isolated vesicles proved to be capable of MCP-methylation in the presence of exogenous Ado- Met, but demethylation required the addition of a cytosolic extract from a wild type strain. When an extract of ache B mutant was used instead of that of the wild-type strain, demethylation was not increased above the control level. These results suggested the che B gene product to be the protein methyl esterase. Labeled vesicles could be used in an assay for esterase activity, whereas the velocity of methylation could be deduced from the incorporation of methyl groups in the absence of cytosolic extract. The activity of the esterase was found to be decreased by attractants and increased by repellents, whereas the opposite effects were noticed for the activity of the methylase. Since normal cells appear to contain only one chemotactic methylase and esterase, the authors suggested receptor-ligand binding to cause a change in the conformation of the respective MCP protein, which would give rise to a changed interaction of the substrate with the cytosolic enzyme system.
Hazelbauer and Harayama (1979), Kondoh et al. (1979) and Hazelbauer et al. (1981) characterized chemotactic mutants of E. coli which do not respond to gradients of ribose and galactose, but which show a normal response to other chemoattractants (pheno- type trg). The mutants contain normal amounts of ribose and galactose receptors, which also have a sugar transport function. The gene product which is lacking in these mutants therefore seems to be a transducer protein situated between the receptors and the flagella. Kondoh et al. (1979) and Hazelbauer et
al. (1981) demonstrated M C P 3 to be the product of the trg gene. Methylation of this protein is stimulated when ribose or galactose bind to their respective receptors, whereas the protein itself seems to be a membrane component for the coupling of the sugar receptors to the propagation system.
In 1980, several research groups demonstrated that M C P I and M C P 2 are methylated at several sites, so that each protein can produce at least four bands during SDS-polyacrylamide gel electrophoresis (Boyd and Simon, 1980; Chelsky and Dahlquist, 1980a; De Franco and Koshland, 1980; Engstrom and Hazelbauer, 1980). The pattern of bands was ana- lyzed before and after addition of attractants, in the presence and absence of methylation inhibitors and after treatment of the protein with dilute alkali. The banding pattern was also compared in wild type cells, che B and che X mutants. Using this experimental approach, it was proved that the different bands represent forms of one protein with a different num- ber of methylated sites. The authors suggested mul- tiple methylation to be the mechanism necessary for adaptation of the cells to different background levels of attractant, an increased attractant concentration giving rise to an increased occupation of methyl acceptor sites. At least four of such sites were demon- strated on M C P 1 and M C P 2, the addition of each new methyl group causing a small increase in electro- phoretic mobility. Chelsky and Dahlquist (1980a) observed three methyl accepting sites on each tryptic fragment of M C P 2.
The interaction of chemotactic gene products has been examined by cross-linking of proteins with dithio-bis-succinimidyl propionate in bacteria which had been infected with hybrid phages. The phages contained specific bacterial chemotaxis genes and the infection therefore caused an enormous over- production by the bacteria of the corresponding chemotaxis proteins. PME was shown to be a multi- mer with non-homologous subunits, whereas PCM seems to be a dimer. M C P I and M C P 2 appeared to be functional tetramers. The product of the che Z gene could be a regulatory protein, since it is a cytosolic component which interacts both with PCM and PME. The che Wgene product also interacts with PME and with another, yet unidentified protein component (Chelsky and Dahlquist, 1980b).
Hedblom and Adler (1980) and Wang and Kosh- land (1980) were first to present evidence that M C P 1 could be the serine chemoreceptor. Tsr mutants were observed to be not only deficient in serine taxis, but also to show much less binding of ~4C-serine to membrane vesicles. The authors demonstrated that binding of labeled serine to wild-type vesicles was inhibited by M C P /-mediated attractants, whereas M C P 2-mediated effectors did not compete for the serine binding sites. On the basis of these obser- vations, they suggested that the serine chemoreceptor and M C P I were probably identical. In other studies, evidence was obtained which indicated M C P 2 to be the aspartate receptor (Slocum and Parkinson, 1983; Stock and Koshland, 1981). Boyd et aL (1983) presented the nucleotide sequence of the tsr gene of E. coli, which demonstrated the tsr product to be a protein which spans the bilayer. The molecule con- tains two notable stretches of hydrophobic amino
What is the function of protein carboxyl methylation? 427
acids (Ile 7-Leu 33 and Asn 185-Iie 214), which are localized within the inner membrane of the cell. The amino-terminal domain bears the serine receptor site and appears to be on the periplasmic surface of the, membrane, whereas the carboxyl terminal domain seems to fold inside the cell and to contain the sites of methylation.
In 1981, it was demonstrated that the banding pattern of the methyl acceptor proteins of E. coli as observed during two dimensional gel electrophoresis could not be completely caused by multiple methyl- ation. Besides methylation, another chemical modification seems to cause heterogeneity of the MCP population (Hazelbauer and Engstrom, 1981). The modification was found to be dependent on the che B gene product (i.e. PME) and to be stimulated by repellents. It causes a small decrease in the iso- electric point of MCP 2 by one or two charge groups. The modification could not be induced by treatment of the protein with base and the modified MCP was also found to be alkali-stable (Rollins and Dahlquist, 1981; Sherrins and Parkinson, 1981). Kehry and Dahlquist (1982b) showed that two modifications are induced sequentially by the che B gene product on two different tryptic peptides of the molecule, whereas the che B-dependent modification allows additional methyl groups to be incorporated into the first tryptic fragment. Recently, Kehry et al. (1983) performed partial amino acid sequence analysis of the modified peptides, which showed the che B-catalyzed reaction to be a deamidation of glutamine residues to methyl-accepting glutamic acid. Additional evidence for the existence of this process has been provided by Boyd et al. (1983), who showed two of the methyl- ation sites of the serine receptor in the tsr gene to be encoded as glutamine.
Methylation of the glutamate residues in bacterial chemotaxis proteins seems not to occur in a random fashion, but to follow a definite order. Springer et al. (1982) have demonstrated that the residues which are methylated under basal conditions differ from those which are esterified after the addition of attractant. When an attractant is removed, the loss of methyl groups occurs primarily from the stimulated in- crement, whereas the methyl esters of the basal level are much less affected. The turnover of the basal methyl groups was found to be 2-3 times slower than that of the stimulated increment and the distributions of the "basal methyl groups" and the "stimulated methyl groups" over the various electrophoretic bands of the molecule were found to be different.
Slonczewski et al. (1982) have examined the effects of a lowering of extracellular pH on methylation levels in E. coli. The steady-state level of methylation of MCP 1 decreased when the external pH was lowered. A similar response was observed when the internal pH was shifted in a more acidic direction by addition of a membrane-permeant acid. MCP 2 showed smaller changes in methylation than MCP 1 in response to changes in pH. A close correlation was observed between the effectivity of weak acids in lowering internal pH (as measured by 31p-NMR), the demethylation of MCP 1 and the induction of repel- lent behavioral responses. The authors suggested that a change in the external pH of the medium causes a change in the internal pH of the cell, which initiates
an excitation process that gives rise to enhanced tumbling. An adaptation process is also activated, which involves the methylation system and which eventually restores the prestimulus tumbling fre- quency.
II.1.2. Other bacteria. A system for reversible methylation of membrane proteins during chemo- taxis not only exists in E. coli, but it has also been demonstrated in many other species of bacteria.
Using methionine auxotrophic mutants, Aswad and Koshland showed as early as 1975 that methi- onine is required for adaptation of Salmonella ty- phimurium cells to chemotactic stimuli. Methionine- starved cells show a very prolonged decrease of tumbling frequency in response to attractants, whereas the normal adaptive behavior can be restored by the addition of exogenous amino acid.
Stock et al. (1981, 1985) have presented evidence that receptor methylation is not the only mechanism underlying adaptation of Salmonella cells during chemotaxis. Although che B cells show a complete lack of the chemotactic PCM (less than 0.04 methyl groups being incorporated per receptor monomer), they can slowly respond and adapt to all chemotactic stimuli. In the complete absence of carboxyl methyl- ation, certain che R mutants show normal chemo- tactic swarming on semi-solid tryptone agar plates. The authors concluded that chemotaxis is affected by at least two interdependent adaptation systems, only one of which involves receptor methylation.
Recombinant DNA techniques have demonstrated that the chemotaxis genes of Salmonella are struc- turally similar and functionally homologous to the corresponding ones of E. coli (DeFranco and Kosh- land, 1981). Therefore, results of E. coli can be generalized to other species of Gram-negative bac- teria. Russo and Koshland (1983) have sequenced the chemotactic aspartate receptor of S. typhimurium. Like the serine receptor of E. coli, the gene product was found to be a transmembrane protein with two notable stretches of hydrophobic amino acids, the amino terminal domain containing the aspartate re- ceptor site, whereas the carboxyl terminal domain contains the sites for methylation. The aspartate receptor of Salmonella and the serine receptor of E coli seem to be very versatile proteins, since their structure has also been shown to contain two trans- membrane ion-channel elements (Kosower, 1983). This opens the possibility that the signal which the receptor transmits might involve the influx or efflux of a cation (Na, K, Ca?) across the inner cell mem- brane (Frere, 1977; Kosower, 1983).
An increased methylation of membrane proteins in response to attractants and a decrease of methylation upon addition of repellents has also been observed in Halobacterium halobium (Bibikov et al., 1982; Schimz, 1982), Pseudomonas aeruginosa (Craven and Montie, 1983) and Spirochaeta auramia (Kathariou and Greenberg, 1983). In Pseudomonas aeruginosa, methionine starvation of a methionine auxotrophic mutant results in a strong decrease of serine chemo- taxis (Craven and Montie, 1983). The life-cycle of the bacterium Caulobacter consists of a mobile flagellar and a sessile stalked stage. Here, PCM and methyl- accepting proteins have been shown to be only
428 AREN VAN WAARDE
present during the locomotion phase (Shaw et al., 1983).
Gram-positive bacteria, like Bacillus subtilis, differ from their Gram-negative counterparts in the fact that their membrane proteins show a decrease of methylation in response to attractants and an in- crease in response to repellents (Ahlgren and Ordal, 1983; Goldman et al., 1982). The methyltransferase from B. subtilis, however, has been shown to be functionally homologous to the corresponding pro- tein of E. coli, which suggests that the methylation sites on the methyl accepting chemotaxis proteins in both groups of organisms are structurally very simi- lar (Burgess-Cassler and Ordal, 1982).
11.2. Eukaryotes
11.2.1 Cellular slime molds. Slime molds of the genus Dictyostelium can, due to the small size of their genome, be considered as very simple Eukaryotes. Because of their simplicity and their interesting life cycle, they are quite extensively used for the study of morphogenesis and chemotaxis.
The life cycle of Dictyostelium discoideum consists of a unicellular and a multicellular stage. During the unicellular (amoeboid) period, the organism lives in the soil and feeds on bacteria, which are probably detected by positive chemotaxis to Colic acid. After exhaustion of the food supply, the amoebae pass an interphase, followed by cell aggregation. The aggre- gate becomes a pseudoplasmodium or "slug", which moves over the substrate and which finally differentiates into a fruiting body, consisting of a stalk with spores embedded in a slime droplet at its top. After dispersal spores germinate and the life cycle starts anew.
Cell aggregation is mediated by chemotaxis to cyclic AMP, which is detected by cell surface recep- tors. After some hours of starvation, certain cells begin to excrete cAMP in a pulsatile manner, whereas the other amoebae move towards these aggregation centers. Background levels of cAMP in the medium are kept low by the action of an extracellular phos- phodiesterase (see for a description of the complete life cycle Loomis, 1982).
In 1979, Mato and Marin-Cao reported that addition of cyclic AMP to starved amoebae of Dictyostelium discoideum gives rise to a transient increase of the methylation of a 120,000 dalton membrane protein. Using an assay method in which the recovery of methyl groups could be expected to be higher, Van Waarde (1982) observed transient incorporation of radioactivity from (3H-methyl)- methionine into four different proteins after adminis- tration of a pulse of chemoattractant, one of these probably being identical to the protein of Mato and Marin-Cao. Similar responses of protein radio- activity were observed in vegetative amoebae of D. discoideum upon the addition of Colic acid and in aggregative amoebae of D. lacteum after adminis- tration of their chemoattractant, monapterin (Van Waarde, 1983). Both the incorporation of radio- activity into protein and the chemotactic response of aggregative amoebae of D. discoideum were found to be diminished by methyltransferase inhibitors, sug- gesting the existence of a functional relationship
between methylation and chemotaxis (Van Waarde and Van Haastert, 1984).
More recent experiments, however, cast severe doubt on the validity of this conclusion. The nature of protein-associated radioactivity was examined by use of standard biochemical techniques (Van Waarde and Van Hoof, 1985). Protein carboxyl methyl esters should show a characteristic dependence of stability on pH, acid media resulting in very long half-lives, whereas alkaline media induce rapid hydrolysis (Paik and Kim, 1980). The radioactivity on proteins of D. discoideum, however, was found to be labile both at alkaline and acidic pH. Because the ester linkage is a covalent bond, protein-bound carboxyl methyl esters should not be displaced after addition of a large amount of unlabeled methionine to the incubation medium. Radioactivity on proteins from D. dis- coideum, however, was found to be released under these conditions. The nature of the released radio- active compound was examined by cation exchange HPLC and in several different mobile phases, the released product co-eluted with authentic methi- onine. It could be separated from S-adenosyl- methionine, decarboxylated S-adenosylmethionine, 5'-methylthioadenosine and methanol. Association of radioactivity to protein was shown not to occur during incubation of cells with radioactive methi- onine, but to take place i,a the cell lysate after treatment of the amoebae with perchloric acid.
On the basis of these experiments, it was concluded that protein-associated radioactivity in cellular slime molds, as measured in the previously mentioned papers, did not represent protein carboxyl methyl- ation, but a reversible association of methionine with certain protein components in the cell lysate. When the lysate is prepared at different periods of time after stimulation of the cells with their chemoattractant, the amount of radioactivity associated with protein shows a time course which closely resembles that of the amount of actin associated with the cytoskeleton. Cells which are stimulated with a large amount of extracellular calcium after treatment with the iono- phore A23187 show a strong, transient increase of the association of methionine to protein, whereas the labeled protein-methionine complex possesses an acidic iso-electric point (Van Waarde and Van Hoof, unpublished results). This evidence suggests that me- thionine associates to the acid-insoluble cytoskeleton.
In the experiments described above, cells were incubated with methionine in the presence of cy- cloheximide, an inhibitor of protein synthesis. Sub- sequently, they were analysed by addition of per- chloric acid (final concentration 6%). After re- suspension in sample buffer, precipitated protein was applied to a polyacrylamide gel and proteins were separated by electrophoresis according to the method of Gagnon et al. (1978b). Finally, the gel was sliced into thin sections and radioactivity in each slice was determined by liquid scintillation counting.
It is also possible to measure protein carboxyl methylation as the amount of radioactive methanol which is formed after treatment of labeled protein with alkali (Diliberto and Axelrod, 1974). Labeled protein is treated with borate buffer of alkaline pH and when hydrolysis of methyl esters is complete, methanol is extracted from the reaction medium with
Gene
What is the function of protein carboxyl methylation?
Table 1. Gene products involved in signal transduction during bacterial chemotaxis
Molecular Phenotype mass of of deletion Nature of Behavior of
product(s) mutant product deletion mutant
che A 77,66kD the B 38kD PME Impaired deadaptation
Impaired demethylation the D 64kD tsr MCP 1 Impaired serine taxis.
(serine receptor) Little serine binding che M 63kD tar MCP 2 Impaired aspartate taxis
(aspartate receptor) and binding che W 12kD Regulatory protein for PME the X 28kD Chemotactic PCM Impaired adaptation to constant
stimuli. Lack of receptor methylation
che Y 8kD Regulatory protein for PCM and PME
che Z 24kD trg MCP 3 Impaired ribose and galactose (sugar signal taxis, but normal sugar binding transducer) and uptake
429
a mixture of toluene and isoamylalcohol. Radio- activity in the organic phase is subsequently deter- mined before and after evaporation. The difference in count rate between the immediately counted and previously evaporated sample represents the volatile fraction of radioactivity, which is assumed to consist o f methanol.
When this assay was applied to the measurement of carboxyl methylation in D. discoideum, it was found that the experimental set-up was very critical.
When the organic phase is evaporated at a tem- perature of 70-90°C, as is the normal procedure of most authors, the methyl group of many organic compounds (including methionine and 5'-methylthioadenosine) is unstable, so that the vol- atile fraction of radioactivity does not necessarily represent only methanol. Data for the temperature stability of authentic methanol are given in Table 2. It is clear that the assay of Diliberto and Axelrod (1974) is most trustworthy if evaporat ion is performed at room temperature and the methylated protein is thoroughly washed before treatment with borate. Therefore, a modified procedure was described which avoids overestimation of the actual methanol pro- duction (Van Waarde and Van Hoof, 1985).
If the modified assay is applied to the measurement of carboxyl methylation during chemotaxis of Di- ctyostelium discoideum, methylation is found to be below the limit of detection during all stages of the life-cycle. This observation makes it unlikely that carboxyl methylation would perform a similar func-
tion during chemotaxis of cellular slime molds as it does in chemosensory perception of enteric bacteria (Van Waarde and Van Hoof, 1985).
II.2.2. Mammalian phagocytic cells. O ' D e a et al. (1978) have presented evidence for a function of protein carboxyl methylation in the chemotaxis of rabbit neutrophils. Stimulation of these cells with the peptide attractant fMet-Leu-Phe causes a transient increase of the incorporation of radioactivity from (3H-methyl)-methionine into protein. Antagonistic peptides were found to block both the chemotactic and methylation responses to fMet-Leu-Phe, al- though they were without effect on basal levels of methylation. Chemotactically inactive cells did not show any stimulus-induced response of methylation, whereas in normal cells, high levels of attractant cause inhibition of both methylation and chemotaxis. When methylation is inhibited by administration of the drug 3-deaza-adenosine, the cells show a concomitant decrease of their chemotactic response to fMet-Leu-Phe. 3-Deaza-adenosine functions as a substrate for S-adenosylhomocysteine hydrolase, but it is not broken down by adenosine deaminase. In neutrophils and macrophages, its administration therefore results in a rapid accumulation of intra- cellular 3-deaza-adenosylhomocysteine, which is a strong inhibitor of protein carboxyl methylase (Chiang et al., 1979; Aksamit et al., 1982). Addit ion of the adenosine deaminase inhibitor E H N A with adenosine and homocysteine thiolactone to the ex- tracellular medium of neutrophils has been shown to
Table 2. Stability of l-(3H-methyl)-methionine at different evaporation temperatures
Temperature (°C) 350 (hot plate) 90 20
Control 100+/-2% Control 100+/-2% Control 100+/-1% dryness 83 after 2h 95 + / - 2% dryness 98 + / - 2% 15s dry 61 overnight 47 + / - 3% 30s dry 32 45s dry 19
Values represent the amounts of radioactivity remaining after evaporation at the temperatures indicated. It is clear that at 90 °, the methyl group of methionine is unstable and released in volatile form, resulting in about 50% loss of activity during one night, whereas at 20 ° even after several weeks no loss of activity occurs. Data from Van Waarde and Van Hoof (1985).
430 AREN VAN WAARDE
cause rapid accumulation of intracellular adenosyl- homocysteine with concomitant decreases in methyl- ation and chemotaxis (Chiang et al., 1979).
Protein carboxyl methylase of leukocytes appears to be specific for certain endogenous membrane proteins. Addition of exogenous methylase from ad- renal gland to a leukocyte homogenate causes a strong increase of the number of methylated proteins. Especially the cytosol appears to contain several potential methyl acceptors which are not methylated by the endogenous PCM (Schiffmann et al., 1979). Methylation seems to be related to calcium move- ments, since addition of 5 mM EGTA inhibits the methylation and chemotactic responses by about 70%, without affecting fMet-Leu-Phe binding to the cell surface receptors (Schiffmann et al., 1979). Both a protein carboxyl methylase and a protein methyl esterase appear to be activated after stimulation with chemoattractant, resulting in an increased turnover of protein methyl esters (Venkatasubramanian et al., 1980).
Drugs which give rise to accumulation of intra- cellular S-adenosylhomocysteine have been shown to cause inhibition of "capping" of murine and human lymphocyte surface immunoglobulin. Normally, lig- ands to surface Ig cause a rapid redistribution of the receptors to one pole of the cell, but in B lymphocytes which have been treated with methylation inhibitors, this response is impaired. The redistribution of recep- tors over the cell surface appears to require trans- methylation (Braun et al., 1980). Since only the capping of surface Ig, but not that of other surface macromolecules is blocked by inhibitors of trans- methylation, the authors suggested that methylation may be required for the triggering of microfilament activity. Surface Ig is able to interact with the con- tractile apparatus, whereas the other surface macro- molecules which have been examined do not couple to the cytoskeleton (Braun et al., 1980). The ability of murine macrophages to lyse P815 tumor cells has been observed to be also decreased by inhibitors of transmethylation. The iysis process is affected at an early stage, since the drugs inhibit macrophage bind- ing to the neoplastic target cell (Adams et al., 1981).
It should be noted that the experiments using drugs cannot be considered as proof of an involvement of methylation reactions in the processes of phago- cytosis and chemotaxis, since their effects on a living cell are not limited to inhibition of protein methyl- ation. 3-Deaza-adenosine has been shown to decrease also the synthesis of a small class of chemotaxis specific proteins in a mouse marophage cell line. When total protein synthesis is inhibited by incu- bation of the cells with cycloheximide, puromycin or actinomycin D, chemotaxis can be inhibited to an equal amount as after administration of 3-deaza-adenosine. Therefore, the effect of deaza- adenosine on macrophage chemotaxis could be re- lated to inhibition of the synthesis of specific proteins rather than to depression of carboxyl methylation (Aksamit et al., 1983). Garcia-Castro et al. (1983) have presented evidence which suggests that there is no direct relationship between methylation and chemotaxis. In rabbit neutrophils, protein carboxyl methylation could be inhibited up to 65% without any influence on chemotaxis. Paradoxical effects of
adenosine were also noted. Whereas adenosine itself had no effect on chemotaxis, it prevented almost completely the inhibition of chemotaxis of neu- trophils subsequently treated with deaza-adenosine. Yet the deaza-adenosine-induced inhibition of car- boxyl methylation was not significantly altered by preincubation with adenosine. This result suggests that the inhibition of chemotaxis by 3-deaza- adenosine might be due not to inhibition of protein methylation, but rather to the blocking of some vital reaction which has yet to be discovered. Sung and Silverstein (1985) have demonstrated that phago- cytosis of macrophages also is not directly related to protein carboxyl methylation. After removal of methylation inhibitors from the extracellular medium by washing, methylation and phagocytosis are re- stored with quite different time constants, phago- cytosis having returned to normal about 90min earlier than carboxyl methylation.
Methylation inhibitors may also interfere with cyclic nucleotide metabolism. In some mammalian cell preparations, accumulation of S-adenosyl- homocysteine and 3-deaza-adenosylhomocysteine has been shown to cause a significant increase of the basal level of cAMP and a potentiation of the hormone-induced response of adenylate cyclase (Zi- mmerman et al., 1978, 1984; Shattil et al., 1982). In Dictyostelium discoideum, homocysteine and homo- cysteine thiolactone cause inhibition of cyclic nucleo- tide phosphodiesterase (Van Waarde and Van Haas- tert, 1986; Van Waarde, 1986).
Discussion of the role of methylation in leukocyte chemotaxis is further complicated by the techniques authors used for the measurement of carboxyl methylation. In some of the earlier papers, the effects of methylation inhibitors on the specific radioactivity of intracellular S-adenosyl-(3H-methyl)-methionine was not examined, so that the data for inhibition of methylation cannot be quantitatively compared to those for inhibition of chemotaxis. Carboxyl methyl- ation is usually measured as the amount of methanol formed from methylated protein in the assay of Diliberto and Axelrod (1974). Since all authors evap- orate the extracted radioactivity at elevated tem- perature, there is a serious possibility of evaporation of labeled compounds besides methanol, so that what is measured in the assay should not necessarily be carboxyl methylation (Van Waarde and Van Hoof, 1985). It is also to be noted that the data of O'Dea et al. (1978b) concerning a transient response of carboxyl methylation in neutrophils to chemo- attractants could not be reproduced in macrophages (Pike et al., 1979; Snyderman and Pike, 1979).
In summary, it can be said that positive evidence for a general involvement of methylation in the directed movement of mammalian chemotactic cells is lacking. In some phagocytic cells, responses of protein carboxyl methylation to chemoattractants could not be observed, whereas the evidence presented to support the existence of such a response in other cell types is open to criticism. In carefully controlled recent experiments, a lack of correlation is observed between carboxyl methylation, phago- cytosis and chemotaxis (Garcia-Castro et al., 1983; Sung and Silverstein, 1985). It is therefore by no means sure that the data obtained on the involvement
What is the function of protein carboxyl methylation? 431
of methylation in chemotaxis of enteric bacteria can be generalized to the regulation of directed movement in Eukaryote cells.
III. CARBOXYL METHYLATION DURING EXOCYTOTIC SECRETION
Protein carboxyl methylation has also been pro- posed to play a role in the coupling of exocytotic secretion to the stimuli of various agonists (Gagnon and Heisler, 1979).
In adrenal medulla, the presence of PCM and methyl acceptor proteins has been demonstrated by Dilibcrto et al. (1976). The enzyme is cytosolic and the methyl accepting protcins are mainly localized in chromaffin vesicles. When thc adrenal medulla is neurogcnically stimulated, the ievcl of mcthylation of these proteins is remarkably increased. The authors suggested that the function of the enzyme could be the neutralization of negative charges on the external surface of the vcsiclc, thereby increasing its proba- bility of collision and fusion with the plasma mem- brane. By this mechanism, cxocytotic secretion would be enhanced.
Borchardt et al. (1978) have attempted to separate chromaffin granule membrane proteins by chro- matography on Biogel A15. The methyl-accepting protein had a similar retention time as dopamin-fl hydroxylase and chromogranin A, which co-eluted on this column. Therefore, the radioactive peak was subjected to SDS-polyacrylamide gel electrophoresis followed by concanavalin A-agarose chro- matography. In the latter system, the methylacceptor and dopamine-/~ hydroxylase could be separated. On the basis of this evidence, the authors concluded that the methylated protein might be chromogranin A. Using an acidic polyacrylamide gel, however, Gag- non et al. (1978b) demonstrated that the radioactive protein is different from chromogranin A. At least four proteins appeared to be labeled after incubation with radioactive methionine: two from the membrane with apparent molecular mass 32,000 and 55,000 and two from the cytosol with molecular mass 48,000 and 63,000. Diliberto et al. (1979) have reported that hypoglycemia induces a remarkable increase of car- boxyl methylation in chromaftin-containing cells,
In rat pancreas, Povilaitis et al. (1981) found a cytosolic PCM, membrane-bound methyl acceptor proteins, cytosolic and microsomal PME. Stimu- lation of pancreatic cells with the hormone pan- creozymin or the cholinergic agonist carbachol in- duces both protein carboxyl methylation and amylase release. The two processes are activated at the same doses of agonist and they follow identical time courses. Receptor antagonists, like dibutyryl-cyclic GMP and atropine, block methylation as well as amylase release. Extracellular EGTA has a similar effect, suggesting calcium influx to be necessary for protein esterification and enzyme secretion. When methylation was inhibited by drugs for more than 70%, however, the response of amylase secretion to agonists was unimpaired. The conclusion that car- boxyl methylation is involved in stimulus-secretion coupling has therefore been criticized by Davison (Davison et al., 1982), who postulated that methyl-
ation and secretion are simultaneously, but indepen- dently activated by the coupling mechanism. It could be argued, however, as Heisler and Gagnon have done (Davison et al., 1982), that PCM catalyzes the methylation of many proteins in different compart- ments of the pancreas, only some of these being involved in exocytosis. Inhibition of total pool- methylation therefore does not necessarily correlate with inhibition of secretion and the hypothesis of an involvement of carboxyl mcthylation in the mech- anism of agonist-induced amylase release has not been falsificd. Hcisler and Lambert (1982) have char- actcrizcd zymogen granule membrane proteins after treatment with purified PCM in vitro. Three poly- pcptidcs were found to be mcthylated with apparent Mr-values of 86,000, 50,600 and 26,600.
Beta-adrcncrgic amylase secretion by rat parotid gland has also been reported to involve carboxyl mcthylation (Strittmattcr et al., 1978). The authors stimulated parotid glands, both in vivo and in vitro, with isoproterenol. This beta-adrenergic agonist seemed to induce amylase secretion and carboxyl methylation. Its effect was blockcd by the beta- adrenergic antagonist propanolol and it could not bc mimickcd by alpha-adrenergic agonists. Thc results of these experiments were subsequently challengcd by Unger et al. (1981) who postulated that the described effects of isoprotcrcnol on methylation were due to expcrimental error. Since Strittmattcr et al. (1978) expressed the levcl of mcthylation as thc number of methyl groups per mg protein, their results wcrc not independent of amylasc release, because release gives rise to a significant reduction of total parotid protein content. Unger et al. (1981) therefore repeated the experiments of thc previous authors and related mcthylation level to DNA instead of protcin content. When the incorporation of methyl groups is thus expressed, stimulation by isoprotercnol is no longer observed. It is therefore unlikely that in parotid gland, carboxyl mcthylation would be involved in coupling of amylase secretion to the beta-adrcnergic receptor.
An increasing amount of evidence suggests how- ever, that carboxyl methylation is involved in neuro- secretion. Diliberto and Axelrod (1974) purified PCM from pituitary gland and examined its substrate specifcity. Hormones from the anterior pituitary (ACTH, FSH, TSH and growth hormone) were found to be good substratcs, whereas posterior pitu- itary hormones (oxytocin, vasopressin) were not methylatcd at all. Methyl acccptors from the pos- terior pituitary were subsequently identified by Kim et al. (1975a), Edgar and Hope (1976) and Diliberto et al. (1976). Kim et al. (1975a) observcd that maximal increases in the level of endogenous methyl acccptor proteins in hypothalamus during organ culture coin- cide with the peak synthcsis of neurophysins. Edgar and Hope (1976) purified PCM from bovine posterior pituitary gland and showed that ncurophysins were indeed the best endogenous methyl acccptors. In thc presence of vasopressin, mcthylation of neurophysin I was found to be increased. Diliberto et al, (1976) also showed that neurophysins were good substrates for PCM and observed an increase of the methylation of neurophysin II in the presence of oxytocin. The work of these authors clearly demonstrated that in
432 ALIEN VAN WAARDE
the anterior pituitary, stored hormones were them- selves the major endogenous methyl acceptors, whereas in the posterior pituitary, hormone carrier proteins served as endogenous substrates. In the latter case, the hormone binding and methyl- ation sites appeared to be not identical. Hormone binding to neurophysin probably induces a con- formational change, which enhances neurophysin methylation.
Kim and Li (1979a,b) showed that ACTH methyl- ation takes place on the Glu 28 residue. Methylation is probably stoichiometric, since levels of 30 mol% of esterification can be reached in vitro. Other hormones like iutropin are also methylated to a large extent. Since increased methylation of prolactin has been shown to be associated with decreased hormonal effectiveness (Diliberto and Axelrod, 1974), the possi- bility exists that methylation is involved in hormone inactivation during storage.
Gagnon et al. (1978a) and Kloog and Saavedra (1983) confirmed the report of previous authors, that methyl acceptors in the posterior pituitary are neuro- physins. The methylated proteins show all the charac- teristics of these carrier proteins, such as apparent Mr of 11,000, disappearance after salt loading or pitu- itary stalk section, labeling by 35S-cysteine and low levels in the homozygous Brattleboro rat. Methyl- ation was found to take place in the hypothalamo- hypophysial nerve endings, which suggests methyl- ation to be involved in hormone release.
Experiments in which the effects of methylation inhibitors on hormone release were examined, have produced promising results. Heisler et aL (1983) have reported that in a mouse pituitary cell line, corticotropin-releasing factor induces ACTH- secretion and protein carboxyl methylation. Both processes were affected with similar time courses and dose-response relationships. Inhibition of methyl- ation with 3-deaza-adenosine and homocysteine thio- lactone caused a decline of ACTH-release, whereas inhibition of ACTH-secretion with dexamethasone gave rise to a decrease of methylation. It is therefore possible that methylation and ACTH secretion in pituitary gland are related events.
Kloog et al. (1983) have postulated that carboxyl methylation could also be involved in the release of neurotransmitters by neuronal cells. When mouse neuroblastoma cells are treated with agents that induce morphological and electrophysiological differentiation, PCM-activity and the amount of membrane-bound methyl acceptor proteins are markedly increased. The time course of methylation closely parallels that of the development of electrical excitability. A treatment of the cells which causes neurite outgrowth without electrophysiological differentiation on the contrary does not induce any PCM-activity and gives rise to only a small increase in methyl acceptor proteins. The authors suggested that PCM may serve to methylate carboxyl groups of ion channel proteins, thereby regulating the perme- ability properties of the membrane.
This interpretation of their data, however, has been weakened by experiments performed by Rabe and McGee (1982). Using a rat neuronal sympathic cell line, which stores noradrenaline and acetylcholine in secretory granules and releases them in response to
K+-depolarization or nicotinic receptor stimulation, these authors demonstrated the turnover of protein methyl esters to be slow as indicated by a pulse-chase experiment. Although the methylation inhibitor 3-deaza-adenosine enhances depolarization- dependent neurotransmitter release (Rabe et al., 1980), protein carboxyl methylation was found to be unaffected by depolarization and inhibition of methylation reached a plateau before enhancement of neurotransmitter release occurred. These results seem incompatible with a function of PCM in a fast regulation mechanism of exocytotic secretion.
It is possible, however, that the coincidence of carboxyl methylation and electrical excitability dur- ing neuronal development is caused by the fact that acetylcholine receptors are the main substrate for protein carboxyl methylase. Methylation of acetyl- choline receptors has been demonstrated in the elec- tric organ of Torpedo cali fornica by Kloog et al. (1980) and Flynn et al. (1982). Purification of methyl acceptor protein from membranes of the electric organ paralleUed enrichment of the acetylcholine receptor. The cytosol of the organ contains a protein carboxyl methylase which is rather specific for the receptor and binds it with a rather high affinity (K m 1 #M). Receptor methylation is not affected by bind- ing of the cholinergic ligand, suggesting the locus of methylation to be different from the acetylcholine binding site. Receptor methylation seems to be stoi- chiometric. Esterification levels up to 6.8 mol% were observed in vitro, but due to low recovery of the methyl esters after polyacrylamide gel electro- phoresis, before separation these can have been as high as 17-45 mol%.
Probably not only acetylcholine receptors, but also benzodiazepine receptor proteins are methylated in the intact brain. After birth, rat brain PCM shows a steady increase in activity during development from 2 to 42 days. When the methyl acceptor substrates are analyzed by gel filtration on Sepharose 4B, carboxyl methylated peaks co-elute with [3H]-flunitrazepam binding activity. Changes in the position and the number of peaks during development are identical in both cases (Gregor and Sellinger, 1983). For this reason, carboxyl methylation may be involved in the maturation of both acetylcholine and benzodiazepine receptor proteins.
It is likely that O-methylation is also involved in the re-uptake of neurotransmitter by adrenergic neu- rons. Membranes from rat cerebral cortex bind S- adenosylhomocysteine with high affinity (0.5 juM). The binding activity does not correspond to any' brain methylase (Fonlupt et al., 1981). Since AdoHcy activates noradrenaline uptake both in vitro and in vivo (Fonlupt et al., 1979), as does adenosine, it is possible that AdoHcy binds to an adenosine receptor. After noradrenaline is released from the pre- synaptic bottom, it is methylated by intersynaptic catechol O-methyltransferase. S-adenosylhomo- cysteine formed in this reaction could be part of a feedback mechanism to regulate neurotransmitter re-uptake. Inhibitors of methylation have been found to decrease also noradrenaline uptake (Samet and Rutledge, 1982), whereas administration of ex- ogenous AdoHcy results in increased uptake (Fon- lupt et al., 1979).
What is the function of protein carboxyl methylation? 433
IV. CARBOXYL METHYLATION DURING ION TRANSPORT
The kidney contains very high levels of protein methyl esterase in comparison to other tissues (Gag- non, 1979). This methyl esterase is not identical to the non-specific carboxylesterase in the same tissue, as is shown by its different sensitivity to inhibitors, different molecular weight and different subcellular localization. Kidney PCM activity is highest in distal tubules and glomeruli, whereas PME is mainly present in proximal tubules (Chene et al., 1982). The authors suggest that methylation could be involved in the regulation of ion transport.
Evidence for such a function of carboxyl methyl- ation has been presented by Wiesmann et al. (1985). In toad bladder cells, aldosterone induces an increase in protein carboxyl methylation, phospholipid methylation and sodium readsorption. Treatment of bladder cells with 3-deaza-adenosine inhibits the basal and aldosterone-stimulated protein carboxyl methylation and completely blocks the aldosterone- induced response of sodium transport. It is not likely that 3-deaza-adenosine exerts its effect via an ade- nosine receptor, since the response of intracellular cAMP to aldosterone was unimpaired. Protein syn- thesis in the bladder cells was also unchanged after treatment with 3-deaza-adenosine. It is therefore possible that carboxyl methylation in kidney and other tissues is related to sodium readsorption.
V. CARBOXYL METHYLATION OF AGED PROTEINS
PCM-activity of mammalian blood is known to be localized mainly in the red ceils (Axelrod and Cohn, 1971, Kim et aL, 1975b). Several authors have there- fore tried to localize and identify erythrocyte methyl accepting proteins. The methyl accepting proteins in erythrocytes appear to be localized mainly in the plasma membrane (O'Dea et al., 1978a). Using SDS- polyacrylamide gel electrophoresis as a separation technique, Galletti et al. (1979a), Kim and GaUetti (1979) and Kim et al. (1980) observed methylation of glycophorin A and "band 4.5", a protein being part of the glucose transport system. Incorporated methyl esters were found to be very labile at neutral and alkaline pH and their formation seemed to require AdoMet as a methyl donor, since it could be inhibited for more than 70% by addition of cycloleucine, a competitive inhibitor of L-methionine S-adenosyl- transferase (Lombardini et aL, 1970; Sufrin, 1979).
Janson and Clarke (1980) have shown that the major site of carboxyl methylation in human erythro- cyte membrane proteins is at aspartyl residues. Using a sophisticated separation method, Freitag and Clarke (1981) and Terwilliger and Clarke (1981) demonstrated incorporation of methyl groups into the band 3 anion transport protein, some cytoskeletal proteins, the major sialoglycoprotein, an unidentified intrinsic polypeptide (Mr 30,000) and an extrinsic polypeptide (Mr 17,000). Changes in the geometry of the membrane may give rise to changes in the turn- over of methyl groups of membrane proteins, as is suggested by comparison of sickle cell with normal erythrocyte carboxyl methylation (Kim et aL, 1982; Green et al., 1983; Ro et al., 1983).
In 1982, Clarke and co-workers showed that the aspartyl sites which are methylated in erythrocyte proteins are in the unusual D-configuration. o-Aspartic acid is not incorporated into proteins during protein synthesis, but D-amino acid residues are spontaneously formed from their L-counterparts during protein ageing (Masters et al., 1977; Friedman and Masters, 1982). For this reason, McFadden and Clarke (1982) postulated that methylation of D-aspartyl groups in erythrocytes could be a step in the repair of aged membrane proteins. Since race- mization of amino acid residues in protein is slow and occurs in a random fashion, methylation at D-aspartyl sites is very substoichiometric (less than 0.02 tool% in erythrocytes).
Later, Barber and Clarke (1983) and Galletti et al. (1983) demonstrated that membrane protein methyl- ation increases indeed with erythrocyte age due to an increase in the number of methylatable sites. Methyl- ation seems to be a general mechanism for the labeling or removal of damaged protein molecules, since very many different erythrocyte proteins have been found to be methylated, including haemoglobin and carbonic anhydrase (O'Connor and Clarke, 1984).
VI. CARBOXYL METHYLATION OF CALCIUM-BINDING PROTEINS
A final area of study with very promising results regarding the function of methylation is the biochem- istry of calcium-binding proteins (Gagnon, 1983). Using a crude preparation of PCM, Cox et al. (1979) demonstrated that calmodulin is a very good sub- strate for protein carboxyl methylase. When the methylation conditions are optimized, up to 50 tool% of methyl groups can be incorporated (Gagnon et al., 1981), so calmodulin methylation appears to be stoi- chiometric. Calcium binding to calmodulin decreases its ability to be methylated in a dose-dependent manner (Gagnon et al., 1981). Other calcium-binding proteins, like troponin C, are also methylated to a greater or lesser extent dependent on the amount of calcium being present (Gagnon, 1982, 1983). Methyl- ation of calmodulin was found to reduce the capacity of calmodulin to activate cyclic nucleotide phos- phodiesterase and the degree of reduction was ob- served to correlate with the extent of methylation (Gagnon et al., 1981). Evidence for methylation of calmodulin in intact cells has been presented by Sitaramayya et al. (1980), Gagnon et al. (1981) and Campillo and Ashcroft (1982). Calmodulin methyl- ation also induces a decreased ability of calmodulin to activate calcium-dependent protein kinase (Bill- ingsley et al., 1983). If methylation of calcium- binding proteins is indeed involved in the regulation of calcium-dependent processes, this could provide an explanation for the often observed correlation between PCM activity and sperm mobility (Bouchard et al., 1980; Cusan et al., 1981; Gagnon et al., 1979, 1982; Gordeladze et al., 1982; Janson and Sastry, 1981; Purvis et aL, 1982).
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