Transport of Sulfonium Compounds

We report here the characterization and the molecular analysis of the two high affinity permeases that mediate the transport of S-adenosylmethionine (AdoMet) andS-methylmethionine (SMM) across the plasma membrane of yeast cells. Mutant cells unable to use AdoMet as a sulfur source were first isolated and demonstrated to lack high affinity AdoMet transport capacities. Functional complementation cloning allowed us to identify the corresponding gene (SAM3), which encodes an integral membrane protein comprising 12 putative membrane spanning regions and belonging to the amino acid permease family. Among amino acid permease members, the closest relative of Sam3p is encoded by theYLL061w open reading frame. Disruption ofYLL061w was shown to specifically lead to cells unable to use SMM as a sulfur source. Accordingly, transport assays demonstrated that YLL061w disruption mutation impaired the high affinity SMM permease, and YLL061w was therefore renamedMMP1. Further study of sam3Δ andmmp1Δ mutant cells showed that in addition to high affinity permeases, both sulfonium compounds are transported into yeast cells by low affinity transport systems that appear to be carrier-facilitated diffusion. We report here the characterization and the molecular analysis of the two high affinity permeases that mediate the transport of S-adenosylmethionine (AdoMet) andS-methylmethionine (SMM) across the plasma membrane of yeast cells. Mutant cells unable to use AdoMet as a sulfur source were first isolated and demonstrated to lack high affinity AdoMet transport capacities. Functional complementation cloning allowed us to identify the corresponding gene (SAM3), which encodes an integral membrane protein comprising 12 putative membrane spanning regions and belonging to the amino acid permease family. Among amino acid permease members, the closest relative of Sam3p is encoded by theYLL061w open reading frame. Disruption ofYLL061w was shown to specifically lead to cells unable to use SMM as a sulfur source. Accordingly, transport assays demonstrated that YLL061w disruption mutation impaired the high affinity SMM permease, and YLL061w was therefore renamedMMP1. Further study of sam3Δ andmmp1Δ mutant cells showed that in addition to high affinity permeases, both sulfonium compounds are transported into yeast cells by low affinity transport systems that appear to be carrier-facilitated diffusion. Transport of sulfonium compounds. Characterization of the S-adenosylmethionine andS-methylmethionine permeases from the yeastSaccharomyces cerevisiae.Journal of Biological ChemistryVol. 274Issue 47PreviewPage 28099, Fig. 5: There were several errors in the presentation of the sequences of Sam3p and Mmp1p, including the inversion of the identifying names. The corrected figure is shown below. This error does not affect the rest of the paper. Full-Text PDF Open Access Among the wide diversity of naturally occurring metabolites that contain a sulfur atom, few are sulfonium salts. In fact, only four sulfonium metabolites have been reported to exist in living organisms. These are S-adenosylmethionine (AdoMet), 1The abbreviations used are:AdoMetS-adenosylmethionineAAPamino acid permeaseSMMS-methylmethionineORFopen reading frame S-methylmethionine (SMM), and their two derivatives, decarboxylated S-adenosylmethionine and dimethylsulfoniopropionate, respectively (Fig. 1 A). The unique property that actually defines these sulfonium compounds is the presence of a trivalent sulfur atom. As a consequence of the electron deficiency at the sulfur pole, the carbon-sulfur bonds that are present in sulfonium compounds are highly susceptible to nucleophilic displacement reactions. The way by which such a chemical reactivity was recruited by living organisms to establish their intermediary metabolism is strikingly exemplified by AdoMet, which is involved in a large set of remarkably versatile reactions. Indeed, all the chemical moieties linked to the sulfur atom of AdoMet can be transferred, with or without modification, to a large number of acceptor molecules. AdoMet serves as donor of its methyl group in the majority of the transmethylation reactions resulting in protein, nucleic acid, lipid, and soluble metabolites modifications. AdoMet further functions as donor of an amino group during the biosynthesis of biotin (1Stoner G.L. Eisenberg M.A. J. Biol. Chem. 1975; 250: 4029-4036Abstract Full Text PDF PubMed Google Scholar), as donor of a carboxyaminopropyl group for nucleotide modifications, and also as donor of an adenosine group during the synthesis of queuine, a modified base present in some tRNAs from Eschericha coli (2Slany R.K. Bosl M. Crain P.F. Kersten H. Biochemistry. 1993; 32: 7811-7817Crossref PubMed Scopus (71) Google Scholar). In addition, AdoMet is the immediate precursor of the sulfonium decarboxylated S-adenosylmethionine, which in turn constitutes the obligate precursor for the biosynthesis of polyamines, spermine, and spermidine (3Balasundaram D. Xie Q.W. Tabor C.W. Tabor H. J. Bacteriol. 1994; 176: 6407-6409Crossref PubMed Google Scholar). The metabolism of SMM was less extensively characterized than that of AdoMet. SMM was first identified as a constituent of cabbage leaves in 1954 (4McRorie R.A. Sutherland G.L. Lewis M.S. Barton A.D. Glazener M.R. Shive W. J. Am. Chem. Soc. 1954; 76: 115-118Crossref Scopus (72) Google Scholar). Since then, this sulfonium compound has been found in a large number of plants (see Ref.5Mudd S.H. Datko A.H. Plant Physiol. 1990; 93: 623-630Crossref PubMed Scopus (76) Google Scholar and references therein). Biosynthesis of this compound in plants was shown to result from the S-methylation of methionine by AdoMet (6Greene R.C. Davis N.B. Biochim. Biophys. Acta. 1960; 43: 360-362Crossref PubMed Scopus (25) Google Scholar), and to date, only two enzymes have been found to utilize SMM in plants: SMM-homocysteine S-methyltransferase, which catalyzes the transfer of a methyl group to homocysteine, yielding two methionine molecules, and SMM hydrolase, which cleaves SMM to dimethylsulfide and homoserine (5Mudd S.H. Datko A.H. Plant Physiol. 1990; 93: 623-630Crossref PubMed Scopus (76) Google Scholar). Despite these studies, no physiological role could be attributed to SMM until it was shown to be the precursor of the biosynthesis of dimethylsulfoniopropionate. This sulfonium compound is accumulated by many marine algae and by the angiosperms Wollastonia biflora and Spartina alterniflora (see Ref. 7Kocsis M.G. Nolte K.D. Rhodes D. Shen T.-L. Gage D.A. Hanson A.D. Plant Physiol. 1998; 117: 273-281Crossref PubMed Scopus (63) Google Scholar and references therein). Dimethylsulfoniopropionate has been shown to function as an osmoprotectant for bacteria (8Jebbar M. Talibart R. Bernard T. Blanco C. Microbiology. 1994; 140: 2415-2422Crossref PubMed Scopus (63) Google Scholar, 9Lever M. Peddie B.A. Chambers S.T. Biochim. Biophys. Acta. 1996; 1291: 189-194Crossref PubMed Scopus (25) Google Scholar), and the accumulation of dimethylsulfoniopropionate to high levels in the cytoplasm of algal cells and in W. biflora chloroplasts supports the idea that it functions also in plants as an osmoprotectant (10Dickson D.M.J. Wyn Jones R.G. Davenport J. Planta. 1980; 150: 158-165Crossref PubMed Scopus (99) Google Scholar, 11Dickson D.M.J. Kirst G.O. Planta. 1986; 167: 536-543Crossref PubMed Scopus (134) Google Scholar, 12Trossat C. Rathinasabapathi B. Weretilnyk E.A. Shen T.-L. Huang Z.-H. Hanson A.D. Plant Physiol. 1998; 116: 165-171Crossref PubMed Scopus (53) Google Scholar). S-adenosylmethionine amino acid permease S-methylmethionine open reading frame Despite the wide occurrence of these sulfonium compounds in metabolic processes, little attention has been paid to the mechanisms responsible for their transport across biological membranes. A mutation impairing AdoMet transport in the yeast Saccharomyces cerevisiae was described several years ago (13Spence K.D. Ferro A.J. Petrotta-Simpson T.F. Salvatore F. Borek E. Zappia V. Williams-Ashman H.G. Schlenk F. The Biochemistry of Adenosylmethionine. Columbia University Press, New York1977: 77-82Google Scholar), but it was without subsequent molecular characterization. To address this point, we devised a specific genetic screen in cerevisiae to specifically the genetic of AdoMet transport in This allowed us to identify the high affinity AdoMet permease to the characterization of a of AdoMet but with a low In addition, the here to the molecular of a permease, of specifically which we to be used by cerevisiae as an sulfur source. The yeast used in this are in yeast genetic and and were used as described in in A Scholar). The is a of sulfur atom H. PubMed Google Scholar). S-adenosylmethionine were from and were from Open in a To screen for unable to use AdoMet as sulfur the was with acid to the cells were for in in the presence of AdoMet, by a in the presence of as described by Scopus (134) Google Scholar). The was used to cells of in the presence of AdoMet. This to which were as sulfur source. The resulting were and for their to in the presence of AdoMet as sulfur source. 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The for AdoMet was at cells in to the of the AdoMet was to a of for the assays of the high affinity AdoMet permease and to a of for the assays of the low affinity AdoMet were for and a was with of and in a was that was during The for SMM was as AdoMet was to a of for the of the high affinity permease and to for the of the low affinity analysis of AdoMet the of AdoMet was from to 1 The was for at at the AdoMet analysis of SMM the was from to that the can an of transport the the the have the as in the of an We have to the and from as The and were used in and C. PubMed Scopus Google Scholar). The cerevisiae that allowed the cloning of the gene was by the of a of from in the of The YLL061w has been identified of To this the YLL061w was by from The was to the and with to the The was to the and with to the were the The were by the and the and into by yielding The of the of PubMed Scopus Google Scholar). To of was in yielding of the of was and by a the The resulting was by and and used to to of the was The disruption was in by (see To of was and by a the The resulting was by by for and The resulting was and used to and to The disruption was by analysis bacteria or such as the yeast a set of its in the presence of a large number of both and sulfur Accordingly, a mutant unable to is of in the presence of or AdoMet D. Biol. PubMed Scopus Google Scholar). In the as shown in methionine synthesis from AdoMet could be the methyl the intermediary of and or the D. Biol. PubMed Scopus Google Scholar). This metabolic that the use of as a in a screen for of in the presence of methionine but not in the presence of AdoMet identify the of the AdoMet The was by acid, and a was used to of in the presence of AdoMet as a methionine source (see cells were were and for their to in the presence of AdoMet. This genetic screen allowed the of two unable to in the presence of AdoMet but to in the presence of homocysteine, or The two mutant and were with a The resulting were and the analysis of the that in both the to in the presence of AdoMet as a showed that the present in the two mutant and the genetic to the genetic of this was than the used by K.D. J. Bacteriol. PubMed Google Scholar). To further the that the isolated mutation impaired the AdoMet transport the mutant was to a and the of a resulting was assays were a specific of sulfur In to cells were unable to use AdoMet as a sulfur source this compound was to a of As cells are for in the presence of methionine as a sulfur source To the we used the of cells to in the presence of AdoMet was by a yeast and cells of in the of and in the presence of AdoMet were Among four were was from these and used to the to to use AdoMet as sulfur source. This a and of the of its that it to a of the of analysis that this only open reading frame with the to a protein of amino To that the to the isolated was in the the and to its corresponding of the A the was with a mutant and the resulting was of the showed that in all the two were unable to use AdoMet as sulfur in to the that were to use AdoMet. This demonstrated that does to the To further the function of its encoded gene was by the gene disruption as described were by analysis of the sam3Δ mutant were As this is unable to in the presence of AdoMet used as the sulfur it is of in the presence of a of AdoMet was to the mutant was to This result us to the of AdoMet in yeast cells. The first genetic and AdoMet transport in cerevisiae were by and K.D. J. Bacteriol. PubMed Google Scholar, T.F. K.D. J. Bacteriol. 1975; PubMed Google Scholar). These reported that AdoMet is transported across the plasma membrane by a transport we the of AdoMet in a a large of AdoMet we that not to be with a a transport As shown in the of AdoMet in the is therefore that AdoMet is by than The we the of two permeases a high affinity AdoMet permease of AdoMet of (see for the of and the a low affinity AdoMet permease of AdoMet of this it be that the are not to the presence of the high affinity The for the high affinity AdoMet permease are to reported for AdoMet by (13Spence K.D. Ferro A.J. Petrotta-Simpson T.F. Salvatore F. Borek E. Zappia V. Williams-Ashman H.G. Schlenk F. The Biochemistry of Adenosylmethionine. Columbia University Press, New York1977: 77-82Google in their first analysis of AdoMet transport in we the of AdoMet in cells that not the In to was with its the of AdoMet in the cells to a that in these the AdoMet is by a unique transport The of AdoMet of (Fig. are to for the low affinity of the cells. These therefore that in AdoMet is actually by two transport of high and low the encoded by the To further that Sam3p is the high affinity AdoMet permease, the two and were by a two resulting were for AdoMet permease AdoMet at to specifically the of the high affinity permease In both the two the specific which was than that of the As these the no AdoMet permease these therefore that gene does the high affinity AdoMet permease cerevisiae presence of the of the high affinity AdoMet affinity transport specific in of transported of in of transported of Open in a The protein from the nucleotide of its corresponding gene has a molecular of and a of The analysis was in the The that the protein 12 spanning A the protein the the protein is to the amino acid permease of The is in addition to of that are all and that all to contain 12 spanning to both and genetic studies, the function of of this has been B. PubMed Scopus Google Scholar). In these were shown to transport amino across the plasma was therefore that the with function of this be also amino acid The of the protein reported here that it is not the Among the of the the protein the closest to Sam3p is the of the a gene by the of the yeast To date, a function has not been to this Sam3p and the of the YLL061w are highly As shown in Fig. the two and analysis of that this protein 12 that are of the high of by and it was to that the low affinity AdoMet permease be by the To such a theYLL061w was by and the corresponding was in cells as as in sam3Δ cells (see The of the two resulting and was As shown in Fig. in the presence of AdoMet used as sulfur the of both not from that of their In a we the of AdoMet in both mutant cells. As reported in the disruption of theYLL061w was without AdoMet the of AdoMet was the in the or in the presence of the in both the and the cells. the high and the high affinity AdoMet permease, the protein not to be involved in AdoMet and does not to the low affinity AdoMet analysis of AdoMet in affinity affinity in of in of not and respectively to the and from (see in of not Open in a and respectively to the and from (see We that such a two could a for compounds. As in the AdoMet of the closest is the sulfonium To it has been reported that yeast cells are to in the presence of SMM used as a sulfur the presence of an SMM-homocysteine methyl was in yeast D.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). We first demonstrated that yeast cells are of of was as sulfur source to the we the of cells for the the As shown in Fig. disruption of the the of the cells in the presence of as sulfur source. This was specific for as we have demonstrated (Fig. that the presence of disruption mutation is without the of yeast cells in the presence of or AdoMet used as sulfur In addition, it was that yeast cells the could also use homocysteine, and as sulfur the of the cells and the by the high affinity AdoMet permease and the that this gene could an SMM permease of To this we to the of SMM transport in as as in mutant To was to Gage D.A. Rhodes D. Nolte K.D. T. A.J. Hanson A.D. PubMed Scopus Google Scholar), and the of the resulting compound was by (see SMM transport in yeast cells was with SMM from to The as for AdoMet, SMM is by two permeases with a high affinity for SMM and the a low affinity permease and and of SMM was in cells that a disruption mutation of the and the resulting to a that in mutant SMM is by a unique transport that to the low affinity SMM permease in cells (Fig. and This was by the cells are of than their the used as sulfur source was to all these both the of two SMM permeases in yeast and encodes the high affinity SMM the YLL061w was to the yeast genetic analysis of S-methylmethionine in affinity affinity in of in of not and respectively to the and from (see in of not Open in a and respectively to the and from (see of AdoMet transport in cerevisiae the of this K.D. J. Bacteriol. PubMed Google Scholar, T.F. K.D. J. Bacteriol. 1975; PubMed Google Scholar). As we demonstrated that yeast cells actually two AdoMet permeases, these could be The analysis of the of the AdoMet high affinity permease to be further the as as the high affinity permeases to the of amino acid permeases, several of which have been shown to large We AdoMet and SMM in cells in the presence of sulfur compounds and amino such that we could specifically the of the high affinity permeases As reported in the that the high affinity AdoMet permease to be a specific Among AdoMet an group in of the methyl group of is the of the high affinity AdoMet permease, AdoMet and SMM are less In the presence of sulfur such as homocysteine, or was without the high affinity AdoMet Sam3p to the of we AdoMet transport in the presence of amino These assays that the two amino and the high affinity AdoMet permease, the less As also reported in the high affinity SMM permease is only by methionine and and to a by AdoMet, and of AdoMet and SMM by the high affinity were at a than the 1 for the AdoMet permease and for the SMM affinity AdoMet affinity SMM not was AdoMet was at for the of the high affinity AdoMet permease, and SMM was at for the of the high affinity SMM The were at a than the 1 for the AdoMet permease and for the SMM not Open in a was AdoMet was at for the of the high affinity AdoMet permease, and SMM was at for the of the high affinity SMM reported demonstrated that AdoMet and SMM transport in cells of the high affinity AdoMet permease and the high affinity SMM permease are with to (see the of of AdoMet (Fig. and SMM (Fig. the of the sam3Δ mutant cells a high of AdoMet to be (Fig. the used was to the of the low affinity This was a first that the is not for to the of the low affinity permeases to AdoMet In addition, at the used to the low affinity transport in the high affinity permeases, AdoMet and SMM is in less than 1 These have been described for by Jones Transport in The of the and Scholar). has been shown to mediate in yeast and was also reported to be responsible for the low affinity transport of J. Bacteriol. PubMed Google Scholar, R. 1993; PubMed Google Scholar, R. M. T. J. Bacteriol. PubMed Google Scholar). this of transport is to be to metabolic Jones Transport in The of the and Scholar), AdoMet and SMM was in and mutant cells in the presence of As shown in transport by the low affinity AdoMet and SMM permeases is to 1 In addition, we the of SMM transport in cells in the presence of 1 A). The that these only the low affinity permease is the high affinity SMM permease by the metabolic These that both AdoMet and SMM high affinity permeases are transport as from their to the members, in both low affinity AdoMet and SMM transport could be diffusion. The we to address is the that the low affinity AdoMet transport and the low affinity SMM transport could be by the In the of the genetic characterization of both AdoMet and SMM low affinity permeases, a could not be were that showed that of the low affinity AdoMet permease by SMM and of the low affinity SMM permease by AdoMet appear to be (Fig. This could be an that the two low affinity permeases are of the low affinity AdoMet and SMM permeases to an of was at 1 affinity low affinity AdoMet and SMM permeases were with and affinity affinity high affinity permeases were with affinity was at 1 The low affinity AdoMet and SMM permeases were with and The high affinity permeases were with Open in a A highly specific genetic screen allowed us to a mutation impairing AdoMet transport across the plasma The isolated mutation a genetic which was The study of AdoMet by as as cells to the that the AdoMet transport was Indeed, to was of the of AdoMet was with the of two permeases that by their affinity as as by their transport assays further demonstrated that the isolated specifically the high affinity AdoMet Accordingly, sam3Δ cells were shown to be unable to in the presence of AdoMet. of the high affinity AdoMet permease was by to be a highly specific transport by only AdoMet such These therefore that both the carboxyaminopropyl group and the adenosine group of AdoMet are by the AdoMet permease, a during the analysis of AdoMet transport T.F. K.D. J. Bacteriol. 1975; PubMed Google Scholar). it be that the amino and AdoMet The genetic characterization of the mutation allowed us to the corresponding which to of The protein to be an integral membrane protein comprising 12 spanning As by B. PubMed Scopus Google Scholar), membrane in transport in yeast can be into several protein to their In such membrane the transport of compounds. at the of this of the amino acid permeases to of highly the family. A to this was by the of the responsible for the of This sulfur amino acid is transported permeases addition to the amino acid two of at are to the of the family. As the chemical of AdoMet is by the addition of a of methionine to an adenosine a have been to the AdoMet permeases to the the transport of or the The analysis of the that it is not the the protein to the of the (Fig. no could be Sam3p and the identified yeast To the of Sam3p is the first report of a protein belonging to the that does not transport of the amino This result the of by the of and genetic studies, the functions that have been the of by be that in the of the yeast the plasma such a was demonstrated by the of the by the the of which is to of the Lewis D.A. Biol. 1993; PubMed Scopus Google Scholar). We were to identify as a SMM permease the protein encoded by the YLL061w a of which with an than to SMM and in not been the presence of a SMM-homocysteine was in yeast D.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In the present we have that SMM is a sulfur source to of cerevisiae cells and that it is transported in the yeast cells by two permeases, with a high affinity and the a low affinity for that the high affinity SMM permease is a specific permease, only by AdoMet, and study that the amino are to with the of the two sulfonium SMM and the AdoMet The low affinity AdoMet and SMM permeases identified by not the of both to a metabolic are processes, the of is This the two low affinity transport systems described here as diffusion. AdoMet and SMM low affinity permeases are the transport is could be an of the presence of only but genetic is to this and methionine has shown that these sulfur compounds are transported by two and transport systems H. D. V. W. PubMed Google Scholar, A.D. D. J. Biol. 1996; PubMed Scopus Google Scholar), the systems in their affinity for the in their transport and in their The reported here that AdoMet and SMM are two sulfur of which is also transported by two transport systems with This is a for yeast cells to their by the to sulfur compounds the We for