Vol. 123, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, August 1975, p. 516-522 Copyright © 1975 American Society for Microbiology
Specificity and Genetics of S-Adenosylmethionine Transport in Saccharomyces cerevisiae T. F. PETROTTA-SIMPSON, J. E. TALMADGE, AND K. D. SPENCE* Department of Bacteriology and Public Health, Washington State University, Pullman, Washington 99163 Received for publication 21 April 1975
The specificity of a transport system for S-adenosylmethionine was determined through the use of structurally related derivatives. Of the compounds tested, the analogues S-adenosylethionine and S-inosylmethionine and the naturally occurring compounds S-adenosyl-(5')-3-methylthiopropylamine and S-adenosylhomocysteine competitively inhibited uptake of the sulfonium compound. Ki values for these compounds indicate that the order of affinity for the transport protein is S-adenosylmethionine S-adenosyl-(5')-3-methylthiopropylamine > S-adenosylethionine > S-inosylmethionine > S-adenosylhomocysteine. S-adenosyl-(2-hydroxy-4-methylthio)butyric acid exerted inhibition of a mixed type. S-inosyl-(2-hydroxy-4-methylthio)butyric acid, S-inosylhomocysteine, and S-ribosylhomocysteine were without effect. On the basis of the inhibition data, the methionine-amino, adenine-amino, and methyl groups were identified as groups important in the binding of S-adenosylmethionine to the transport protein. Comparison is made with the specificities of various transmethylating enzymes utilizing S-adenosylmethionine. In addition, a number of conventional and temperature-sensitive S-adenosylmethionine transport mutants were isolated and analyzed in an attempt to identify the structural character of the specific transport protein(s). The data obtained suggest that only a single gene (a single polypeptide) is involved in specific S-adenosylmethionine transport. Apparent interallelic complementation supports the assumption that the functional form of the protein is composed of two or more copies of a monomer.
Various patterns of the specificity of membrane transport in microbial eucaryotes have been described: general or relatively nonspecific permeases that transport several structurally unrelated compounds (1, 7, 16); permeases that exhibit a more narrow specificity catalyzing the uptake of a family of structurally similar compounds (2, 4, 9, 16, 17, 18); and several highly specific permeases which transport only a single compound (5, 6, 8). Active transport has also been shown to occur across the vacuolar membrane of yeast (14, 15). Mutations in both general and specific systems have been isolated (5, 6, 7, 8, 23, 24, 25). In earlier reports from this laboratory, a relatively specific transport system for the sulfonium compound S-adenosylmethionine in Saccharomyces cerevisiae was described (13, 24). Kinetic data and studies with specific transport mutants showed that S-adenosylmethionine and S-adenosylhomocysteine share a single uptake system (13). Four stereoisomers of the natural (- )-S-adenosylmethionine are apparently transported across the cytoplasmic membrane by this same system 516
(15). The toxic analogue S-adenosylethionine competitively inhibited uptake of S-adenosylmethionine while several other smaller structurally related compounds including methionine and adenosine were transported by different systems (13). In this report we describe our current investigations of the specificity and functional nature of the S-adenosylmethionine transport system. Chemically and enzymatically prepared derivatives of S-adenosylmethionine have been employed in order to characterize the specific binding sites on the transport protein. In addition, mutants have been employed to partially analyze the genetic and structural nature of the S-adenosylmethionine transport system. MATERIALS AND METHODS Organism, growth, and uptake. Saccharomyces cerevisiae strain 3701B, a haploid auxotroph requiring uracil, was employed in the uptake specificity studies. The cells were grown at 30 C in medium B (24) plus 0.2 mM uracil on a rotary shaker at 300 rpm. The level of growth was monitored on a Klett-Summerson photoelectric colorimeter (400 to 465 nm). Experi-
VOL. 123, 1975
TRANSPORT OF S-ADENOSYLMETHIONINE
517
Hydrolytic derivatives. S-Ribosyl-L-methionine ments on the uptake of S-adenosylmethionine were performed during exponential phase of growth. After was prepared by the method of Schlenk and Zydek four generations in medium B, cells in mid-log phase (21) involving the alkaline hydrolysis of S-adenosylwere transferred to fresh medium, grown for four more methionine at low temperature. Separation of the hydrolytic products S-ribosylmethionine and adenine log generations and sampled for uptake. Measurements of the initial velocity of entrance of was accomplished by paper chromatography. The S-adenosylmethionine were determined according to hydrolysate was spread in a narrow band on Whatthe method of Murphy and Spence (13). The uptake man No. 1 paper and developed in a solvent system velocity remains linear for only approximately the containing 1 liter of 0.1 M sodium phosphate, pH 6.8, first 4 min at saturation in wild-type strains. The plus 20 ml of 2-propanol. R, values for the principal inhibitory effect of the various derivatives was mea- compounds are S-adenosylmethionine (0.80), Ssured by including the unlabeled compound in the ribosylmethionine (0.95), and adenine (0.40). The uptake medium and noting the reduction in initial desired band was cut out, eluted in water at 4 C, velocity of uptake of labeled S-adenosylmethionine. concentrated by evaporation under reduced pressure, The growth and uptake in experiments involving and stored in the frozen state. the temperature-sensitive mutants was performed in S-ribosyl-L-homocysteine was obtained by the hythe same manner as that employed in the specificity drolysis of S-adenosylhomocysteine with acid at 100 C studies, except that growth of the cells was either at as described by Duerre (3). The hydrolytic fragments 23 or 36 C, and uptake was measured at 36 rather S-ribosylhomocysteine and adenine were separated than 30 C. The intracellular concentration of S- from residual S-adenosylhomocysteine by paper chroadenosylhomocysteine and S-adenosylmethionine of matography as described above. R, values for Sthese cells was determined according to the technique adenosylhomocysteine and S-ribosylhomocysteine are of Shapiro and Ehninger (22). 0.62 and 0.87, respectively. For the determination of S-ribosylmethionine and Compounds. S-Adenosyl-L-methionine and Sadenosyl-L-homocysteine were obtained from Boeh- S-ribosylhomocysteine, which lack the ultravioletringer-Mannheim Corp.; [" CH ,]S-adenosyl-L-methi- absorbing moiety, pentose was measured by the onine and L- ["CHs]methionine were from ICN modified orcinol method (21) and sulfur in the Pharmaceuticals; [2-l4C]uracil was from New Eng- thioether linkage by the method of McCarthy and land Nuclear; [8- l4C ladenosine was from Calbio- Sullivan (11). Identities of the compounds were determined on chem; and [14COOH]S-adenosyl-L-methionine was from Amersham-Searle. S-adenosyl-L-ethionine was the basis of their responses on thin-layer and/or paper prepared in this laboratory. Additional S-adenosyl- chromatograms (29) to one or more of the following L-homocysteine was also produced by enzymatic procedures: ultraviolet quenching to establish the synthesis from adenosine and L-homocysteine, and presence of a purine moiety; lack of response to ninhydrin spray to indicate the absence of the methioisolated by ion-exchange chromatography (22). Radioactivity was determined in a Nuclear- nine amino group; and positive response to platinum Chicago Unilux III liquid scintillation counter. The iodide spray (28) to indicate intactness of the scintillation fluid was a mixture of toluene: Triton thioether linkage. Spectrophotometry was used to X-100 (2:1), plus 6 g of 2,5-diphenyloxazole and 0.01 g distinguish between adenosine and inosine derivatives of 1,4-bis(5-phenyl-oxazolyl)-benzene per liter. (29). The identities of the compounds were further Sulfonium derivatives. The synthesis and purifi- characterized by hydrolysis to and chromatography of cation of S-inosyl-L-methionine was accomplished known fragments (19, 29). The structures of the according to the directions of Zappia et al. (29). derivatives are shown in Fig. 1. Selection and genetics of transport mutants. S-adenosylhomocysteine was enzymatically deaminated to S-inosylhomocysteine, which was either Three different techniques were employed to isolate isolated at this point or converted to the desired mutants that lack the ability to transport S-adenosylsulfonium compound by methylation with methyl methionine. The samP locus has been previously designated ai and sam-p (24). The samP3 mutation iodide. S-adenosyl-L-(2-hydroxy-4-methylthio)butyric acid was isolated by random assay of ultraviolet treated was prepared with nitrous acid under conditions cells (24). Seven transport mutants were isolated by appropriate for the removal of the methionine amino selecting strains resistant to S-adenosylethionine. group (29). Separation from other reaction products These organisms were selected from haploid strain 83384-M2 (a ade) after ultraviolet treatment of the was accomplished on ion-exchange columns (29). Deamination of S-adenosylmethionine with nitrous parental strain as previously described (24). The acid under more rigorous conditions (29) resulted in treated cells were plated on medium B containing 0.1 the formation of the doubly deaminated derivative, mM S-adenosylethionine plus 0.1 mM adenine. The parental strain is inhibited under these conditions. S-inosyl-L-(2-hydroxy-4-methylthio)butyric acid. S-adenosyl-(5')-3-methylthiopropylamine was ob- S-adenosylmethionine transport mutants are unaftained by the decarboxylation of S-adenosylmethio- fected by the S-adenosylethionine and may be isonine (29) with the specific enzyme prepared from lated in large numbers. A third type, temperatureextracts of Escherichia coli W (26). After removal of sensitive S-adenosylmethionine transport mutants, the enzyme, the two sulfonium compounds were was isolated from haploid strain 83384-M2 (a ade) separated by an acetic acid gradient (26) on an after exposure to N-methyl-N-nitro-N-nitrosoguanidine. Nitrosoguanidine was added to a 5-h culture Amberlite IRC-50 column.
J. BACTERIOL.
PETROTTA-SIMPSON, TALMADGE, AND SPENCE
518
CH2- S - R3
R
R2
JOURNAL OF BACTERIOLOGY, August 1975, p. 516-522 Copyright © 1975 American Society for Microbiology
Specificity and Genetics of S-Adenosylmethionine Transport in Saccharomyces cerevisiae T. F. PETROTTA-SIMPSON, J. E. TALMADGE, AND K. D. SPENCE* Department of Bacteriology and Public Health, Washington State University, Pullman, Washington 99163 Received for publication 21 April 1975
The specificity of a transport system for S-adenosylmethionine was determined through the use of structurally related derivatives. Of the compounds tested, the analogues S-adenosylethionine and S-inosylmethionine and the naturally occurring compounds S-adenosyl-(5')-3-methylthiopropylamine and S-adenosylhomocysteine competitively inhibited uptake of the sulfonium compound. Ki values for these compounds indicate that the order of affinity for the transport protein is S-adenosylmethionine S-adenosyl-(5')-3-methylthiopropylamine > S-adenosylethionine > S-inosylmethionine > S-adenosylhomocysteine. S-adenosyl-(2-hydroxy-4-methylthio)butyric acid exerted inhibition of a mixed type. S-inosyl-(2-hydroxy-4-methylthio)butyric acid, S-inosylhomocysteine, and S-ribosylhomocysteine were without effect. On the basis of the inhibition data, the methionine-amino, adenine-amino, and methyl groups were identified as groups important in the binding of S-adenosylmethionine to the transport protein. Comparison is made with the specificities of various transmethylating enzymes utilizing S-adenosylmethionine. In addition, a number of conventional and temperature-sensitive S-adenosylmethionine transport mutants were isolated and analyzed in an attempt to identify the structural character of the specific transport protein(s). The data obtained suggest that only a single gene (a single polypeptide) is involved in specific S-adenosylmethionine transport. Apparent interallelic complementation supports the assumption that the functional form of the protein is composed of two or more copies of a monomer.
Various patterns of the specificity of membrane transport in microbial eucaryotes have been described: general or relatively nonspecific permeases that transport several structurally unrelated compounds (1, 7, 16); permeases that exhibit a more narrow specificity catalyzing the uptake of a family of structurally similar compounds (2, 4, 9, 16, 17, 18); and several highly specific permeases which transport only a single compound (5, 6, 8). Active transport has also been shown to occur across the vacuolar membrane of yeast (14, 15). Mutations in both general and specific systems have been isolated (5, 6, 7, 8, 23, 24, 25). In earlier reports from this laboratory, a relatively specific transport system for the sulfonium compound S-adenosylmethionine in Saccharomyces cerevisiae was described (13, 24). Kinetic data and studies with specific transport mutants showed that S-adenosylmethionine and S-adenosylhomocysteine share a single uptake system (13). Four stereoisomers of the natural (- )-S-adenosylmethionine are apparently transported across the cytoplasmic membrane by this same system 516
(15). The toxic analogue S-adenosylethionine competitively inhibited uptake of S-adenosylmethionine while several other smaller structurally related compounds including methionine and adenosine were transported by different systems (13). In this report we describe our current investigations of the specificity and functional nature of the S-adenosylmethionine transport system. Chemically and enzymatically prepared derivatives of S-adenosylmethionine have been employed in order to characterize the specific binding sites on the transport protein. In addition, mutants have been employed to partially analyze the genetic and structural nature of the S-adenosylmethionine transport system. MATERIALS AND METHODS Organism, growth, and uptake. Saccharomyces cerevisiae strain 3701B, a haploid auxotroph requiring uracil, was employed in the uptake specificity studies. The cells were grown at 30 C in medium B (24) plus 0.2 mM uracil on a rotary shaker at 300 rpm. The level of growth was monitored on a Klett-Summerson photoelectric colorimeter (400 to 465 nm). Experi-
VOL. 123, 1975
TRANSPORT OF S-ADENOSYLMETHIONINE
517
Hydrolytic derivatives. S-Ribosyl-L-methionine ments on the uptake of S-adenosylmethionine were performed during exponential phase of growth. After was prepared by the method of Schlenk and Zydek four generations in medium B, cells in mid-log phase (21) involving the alkaline hydrolysis of S-adenosylwere transferred to fresh medium, grown for four more methionine at low temperature. Separation of the hydrolytic products S-ribosylmethionine and adenine log generations and sampled for uptake. Measurements of the initial velocity of entrance of was accomplished by paper chromatography. The S-adenosylmethionine were determined according to hydrolysate was spread in a narrow band on Whatthe method of Murphy and Spence (13). The uptake man No. 1 paper and developed in a solvent system velocity remains linear for only approximately the containing 1 liter of 0.1 M sodium phosphate, pH 6.8, first 4 min at saturation in wild-type strains. The plus 20 ml of 2-propanol. R, values for the principal inhibitory effect of the various derivatives was mea- compounds are S-adenosylmethionine (0.80), Ssured by including the unlabeled compound in the ribosylmethionine (0.95), and adenine (0.40). The uptake medium and noting the reduction in initial desired band was cut out, eluted in water at 4 C, velocity of uptake of labeled S-adenosylmethionine. concentrated by evaporation under reduced pressure, The growth and uptake in experiments involving and stored in the frozen state. the temperature-sensitive mutants was performed in S-ribosyl-L-homocysteine was obtained by the hythe same manner as that employed in the specificity drolysis of S-adenosylhomocysteine with acid at 100 C studies, except that growth of the cells was either at as described by Duerre (3). The hydrolytic fragments 23 or 36 C, and uptake was measured at 36 rather S-ribosylhomocysteine and adenine were separated than 30 C. The intracellular concentration of S- from residual S-adenosylhomocysteine by paper chroadenosylhomocysteine and S-adenosylmethionine of matography as described above. R, values for Sthese cells was determined according to the technique adenosylhomocysteine and S-ribosylhomocysteine are of Shapiro and Ehninger (22). 0.62 and 0.87, respectively. For the determination of S-ribosylmethionine and Compounds. S-Adenosyl-L-methionine and Sadenosyl-L-homocysteine were obtained from Boeh- S-ribosylhomocysteine, which lack the ultravioletringer-Mannheim Corp.; [" CH ,]S-adenosyl-L-methi- absorbing moiety, pentose was measured by the onine and L- ["CHs]methionine were from ICN modified orcinol method (21) and sulfur in the Pharmaceuticals; [2-l4C]uracil was from New Eng- thioether linkage by the method of McCarthy and land Nuclear; [8- l4C ladenosine was from Calbio- Sullivan (11). Identities of the compounds were determined on chem; and [14COOH]S-adenosyl-L-methionine was from Amersham-Searle. S-adenosyl-L-ethionine was the basis of their responses on thin-layer and/or paper prepared in this laboratory. Additional S-adenosyl- chromatograms (29) to one or more of the following L-homocysteine was also produced by enzymatic procedures: ultraviolet quenching to establish the synthesis from adenosine and L-homocysteine, and presence of a purine moiety; lack of response to ninhydrin spray to indicate the absence of the methioisolated by ion-exchange chromatography (22). Radioactivity was determined in a Nuclear- nine amino group; and positive response to platinum Chicago Unilux III liquid scintillation counter. The iodide spray (28) to indicate intactness of the scintillation fluid was a mixture of toluene: Triton thioether linkage. Spectrophotometry was used to X-100 (2:1), plus 6 g of 2,5-diphenyloxazole and 0.01 g distinguish between adenosine and inosine derivatives of 1,4-bis(5-phenyl-oxazolyl)-benzene per liter. (29). The identities of the compounds were further Sulfonium derivatives. The synthesis and purifi- characterized by hydrolysis to and chromatography of cation of S-inosyl-L-methionine was accomplished known fragments (19, 29). The structures of the according to the directions of Zappia et al. (29). derivatives are shown in Fig. 1. Selection and genetics of transport mutants. S-adenosylhomocysteine was enzymatically deaminated to S-inosylhomocysteine, which was either Three different techniques were employed to isolate isolated at this point or converted to the desired mutants that lack the ability to transport S-adenosylsulfonium compound by methylation with methyl methionine. The samP locus has been previously designated ai and sam-p (24). The samP3 mutation iodide. S-adenosyl-L-(2-hydroxy-4-methylthio)butyric acid was isolated by random assay of ultraviolet treated was prepared with nitrous acid under conditions cells (24). Seven transport mutants were isolated by appropriate for the removal of the methionine amino selecting strains resistant to S-adenosylethionine. group (29). Separation from other reaction products These organisms were selected from haploid strain 83384-M2 (a ade) after ultraviolet treatment of the was accomplished on ion-exchange columns (29). Deamination of S-adenosylmethionine with nitrous parental strain as previously described (24). The acid under more rigorous conditions (29) resulted in treated cells were plated on medium B containing 0.1 the formation of the doubly deaminated derivative, mM S-adenosylethionine plus 0.1 mM adenine. The parental strain is inhibited under these conditions. S-inosyl-L-(2-hydroxy-4-methylthio)butyric acid. S-adenosyl-(5')-3-methylthiopropylamine was ob- S-adenosylmethionine transport mutants are unaftained by the decarboxylation of S-adenosylmethio- fected by the S-adenosylethionine and may be isonine (29) with the specific enzyme prepared from lated in large numbers. A third type, temperatureextracts of Escherichia coli W (26). After removal of sensitive S-adenosylmethionine transport mutants, the enzyme, the two sulfonium compounds were was isolated from haploid strain 83384-M2 (a ade) separated by an acetic acid gradient (26) on an after exposure to N-methyl-N-nitro-N-nitrosoguanidine. Nitrosoguanidine was added to a 5-h culture Amberlite IRC-50 column.
J. BACTERIOL.
PETROTTA-SIMPSON, TALMADGE, AND SPENCE
518
CH2- S - R3
R
R2
