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• W2043640575 abstract "We have identified an oligopeptide transporter in the yeast Saccharomyces cerevisiae which mediates the uptake of tetra- and pentapeptides, including the endogenous opioids leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) and methionine enkephalin (Tyr-Gly-Gly-Phe-Met). The transporter is encoded by the geneOPT1. Yeast expressing OPT1 can utilize enkephalins to satisfy amino acid auxotrophic requirements for growth. The transport of radiolabeled leucine enkephalin exhibits saturable kinetics, with a K m of 310 μm. Transport activity is optimum at acidic pH and sensitive to reagents which uncouple oxidative phosphorylation, suggesting an energy dependence on the proton gradient. Growth, transport, and chromatographic data indicate that leucine enkephalin is not hydrolyzed in the extracellular medium and as such is translocated intact across the cell membrane. The system is specific for tetra- and pentapeptides and can be inhibited by the opioid receptor antagonists naloxone and naltrexone. To date, this is the first example of a eukaryotic transport system which can use enkephalins as a substrate, opening the possibility that a homologue exists in higher eukaryotes. We have identified an oligopeptide transporter in the yeast Saccharomyces cerevisiae which mediates the uptake of tetra- and pentapeptides, including the endogenous opioids leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) and methionine enkephalin (Tyr-Gly-Gly-Phe-Met). The transporter is encoded by the geneOPT1. Yeast expressing OPT1 can utilize enkephalins to satisfy amino acid auxotrophic requirements for growth. The transport of radiolabeled leucine enkephalin exhibits saturable kinetics, with a K m of 310 μm. Transport activity is optimum at acidic pH and sensitive to reagents which uncouple oxidative phosphorylation, suggesting an energy dependence on the proton gradient. Growth, transport, and chromatographic data indicate that leucine enkephalin is not hydrolyzed in the extracellular medium and as such is translocated intact across the cell membrane. The system is specific for tetra- and pentapeptides and can be inhibited by the opioid receptor antagonists naloxone and naltrexone. To date, this is the first example of a eukaryotic transport system which can use enkephalins as a substrate, opening the possibility that a homologue exists in higher eukaryotes. oligopeptide transport leucine enkephalin methionine enkephalin carbonyl cyanide 3-chlorophenylhydrazone 4-(chloromercuri)benzenesulfonic acid Tyr-d-Ala-Gly-Phe-d-Leu Tyr-d-Penicillamine-Gly-Phe-d-Penicillamine tyrosine melanocyte-stimulating hormone inhibitory factor 1 open reading frame Small peptides containing four to five amino acid residues are transported by a recently identified class of peptide transporters named the OPT1 family (1.Lubkowitz M.A. Hauser L. Breslav M. Naider F. Becker J.M. Microbiology. 1997; 143: 387-396Crossref PubMed Scopus (79) Google Scholar, 2.Lubkowitz M.A. Barnes D. Breslav M. Burchfield A. Naider F. Becker J.M. Mol. Microbiol. 1998; 28: 729-741Crossref PubMed Scopus (71) Google Scholar). The amino acid sequence of this family is distinct from that of the PTR family, a ubiquitous group of proton-coupled transporters which selectively transports di- and tripeptides (3.Steiner H.-Y. Naider F. Becker J.M. Mol. Microbiol. 1995; 16: 825-834Crossref PubMed Scopus (202) Google Scholar, 4.Fei Y.J. Ganapathy V. Leibach F.H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 239-261Crossref PubMed Scopus (77) Google Scholar). Phylogenetic analysis suggests that the OPT family is also distinct from the major facilitator superfamily (MFS), a diverse collection of proteins which catalyzes the transport of a wide variety of substrates, including sugars, amino acids, neurotransmitters, and drugs (5.Saier M.H. Adv. Microb. Physiol. 1998; 40: 81-136Crossref PubMed Google Scholar).Members of the OPT family have been identified and characterized in the yeasts Candida albicans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Additional members exist in plants, as indicated by searches of publicly accessible data bases. In mammalian tissues, reports in the literature suggest that the enkephalins, endogenous pentapeptides involved in analgesia in the central nervous system, are transported across the blood-brain barrier by a specific, saturable transport system (6.Banks W.A. Kastin A.J. Am. J. Physiol. 1990; 259: E1-E10PubMed Google Scholar). The existence of enkephalin transporters has been inferred from data obtained by measuring whole brain flux of the peptides in rodents (7.Banks W.A. Kastin A.J. Alcohol. 1997; 14: 237-245Crossref PubMed Scopus (24) Google Scholar, 8.Plotkin S.R. Banks W.A. Waguespack P.J. Kastin A.J J. Neurosci. Res. 1997; 48: 273-280Crossref PubMed Scopus (14) Google Scholar, 9.Slokovic B.V. Mackic J.B. Djuricic B.M. Davson H. J. Neurochem. 1989; 53: 1333-1340Crossref PubMed Scopus (81) Google Scholar, 10.Egleton R.D. Abruscato T.J. Thomas S.A. Davis T.P. J. Pharm. Sci. 1998; 87: 1433-1439Abstract Full Text PDF PubMed Scopus (70) Google Scholar). To date, no protein has been identified in eukaryotes as the discrete enkephalin carrier.In this paper, we report that the endogenous opioids Met-enkephalin and Leu-enkephalin, pentapeptides of amino acid sequence YGGFM and YGGFL, respectively, can be transported by cells expressing the S. cerevisiae ORF YJL212C. When expressed under the control of a constitutive promoter in a high copy number vector, this OPT family member is necessary and sufficient to transport Leu-enkephalin into yeast cells. This is the first example of a genetically defined eukaryotic transport protein which can transport enkephalins across the cell membrane. In accordance with the standard nomenclature forS. cerevisiae, we propose the name OPT1 for this gene.DISCUSSIONIn this paper we assign a function to the previously unknown open reading frame YJL212C in the yeast S. cerevisiae and have named this gene OPT1. The protein encoded by OPT1consists of 799 amino acids, and based on the amino acid sequence the predicted protein structure suggests an integral membrane protein containing 12–14 putative membrane-spanning domains. In addition, the protein contains several motifs unique to the OPT family, the largest of which consists of 10 invariable residues (SPYXEVRXXVXXXDDP) located before the first hydrophobic domain (2.Lubkowitz M.A. Barnes D. Breslav M. Burchfield A. Naider F. Becker J.M. Mol. Microbiol. 1998; 28: 729-741Crossref PubMed Scopus (71) Google Scholar). In this study we have confirmed thatOPT1, like other members of the OPT family, encodes a functional oligopeptide transporter.Because Opt1p exhibited all the molecular characteristics of an OPT family member, it was hypothesized that this protein was an oligopeptide transporter, even though it was known that S. cerevisiae could not utilize any tetra- or pentapeptides tested to date to satisfy auxotrophic requirements under routine growth conditions (1.Lubkowitz M.A. Hauser L. Breslav M. Naider F. Becker J.M. Microbiology. 1997; 143: 387-396Crossref PubMed Scopus (79) Google Scholar, 19.Becker J.M. Naider F. Taylor M.D. Amidon G.L. Peptide-based Drug Design: Controlling Transport and Metabolism. American Chemical Society, Washington D. C.1995: 369-384Google Scholar). To see activity of Opt1, it was necessary to express OPT1 under the control of the ADH promoter, a strong, constitutive promoter which would presumably result in high expression of the gene product. In prior studies, Northern blot analysis confirmed that OPT1 was not expressed at detectable levels under routine conditions of logarithmic growth (2.Lubkowitz M.A. Barnes D. Breslav M. Burchfield A. Naider F. Becker J.M. Mol. Microbiol. 1998; 28: 729-741Crossref PubMed Scopus (71) Google Scholar). These results were independently confirmed by serial analysis of gene expression (SAGE) (20.Velculescu V.E. Zhang L. Zhou W. Vogelstein J. Basrai M.A. Bassett Jr., D.E. Heiter P. Vogelstein B. Kinzler K.W. Cell. 1997; 88: 243-251Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar) which revealed that OPT1 is only expressed at a low level (∼1 copy per cell) following nocodazole arrest in the G2/M phase of the cell cycle. Additional analysis of sporulating yeast cells by DNA microarray analysis indicated that OPT1 was expressed during the late stages of sporulation (21.Chu S. DeRisi J. Eisen M. Mulholland J. Botstein D. Brown P.O. Herskowitz I. Science. 1998; 282: 699-705Crossref PubMed Scopus (1510) Google Scholar). In light of these observations, OPT1 gene expression had to be ectopically induced under the control of a heterologous promoter to enable study of Opt1p function in log phase cells.The product of OPT1 is the oligopeptide transporter Opt1p, which translocates pentapeptides, including both Met- and Leu-enkephalin. In BY4730, a strain of S. cerevisiaeauxotrophic for leucine and methionine, only cells expressingOPT1 could grow on Leu-enkephalin in the absence of exogenous leucine. This indicates that enkephalins are transported intact into the cell and then hydrolyzed. If oligopeptides were hydrolyzed by an extracellular protease prior to transport, then the isogenic control strain (BY4730 transformed with the empty vector pDB20), as well as yeast cells transformed with plasmids encoding other OPT family members (CaOPT1, YPR194C) should be able to utilize the hydrolysis products for growth. Chromatographic analysis supports this postulate; no evidence for degraded forms of Leu-enkephalin could be found in the extracellular medium. In addition, a large body of work exists which demonstrates that di- and tripeptides enter the cell intact and are then rapidly hydrolyzed by intracellular peptidases (19.Becker J.M. Naider F. Taylor M.D. Amidon G.L. Peptide-based Drug Design: Controlling Transport and Metabolism. American Chemical Society, Washington D. C.1995: 369-384Google Scholar).Transport of Leu-enkephalin is pH- and temperature-dependent, suggesting that this is a proton-coupled, energy-dependent process. These observations are supported by the sensitivity of the transporter to agents which disrupt the proton gradient or deplete intracellular ATP. Utilization of the transmembrane proton gradient to energize active transport has been demonstrated for the PTR family of di- and tripeptide transporters (4.Fei Y.J. Ganapathy V. Leibach F.H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 239-261Crossref PubMed Scopus (77) Google Scholar). Uptake of radiolabeled Leu-enkephalin was inhibited in the presence of excess unlabeled Met- or Leu-enkephalin; amidated Leu-enkephalin was an ineffective competitor. Tyr-MIF-1 is an amidated tetrapeptide with opiate and anti-opiate activity. This peptide is a substrate for the previously described blood-brain barrier PTS-1 enkephalin transport activity (6.Banks W.A. Kastin A.J. Am. J. Physiol. 1990; 259: E1-E10PubMed Google Scholar) but, like the amidated form of authentic Leu-enkephalin, was not an effective competitor for yeast Opt1p. This observation is consistent with the need for a free carboxyl terminus for substrate recognition by Opt1p. Tetrapeptides were effective inhibitors, with Lys-Leu-Gly-Leu and des-Tyr1 Leu-enkephalin (Gly-Gly-Phe-Leu) eliminating over 50% of radiolabeled enkephalin accumulation, suggesting that an amino-terminal tyrosine is not essential for substrate recognition. Neither the tripeptide enkephalin fragment Gly-Gly-Phe nor the dipeptide Leu-Leu could inhibit uptake, indicating that this system is distinct from Ptr2p and is selective for tetra- and pentapeptides. These data suggest that intact oligopeptides are gaining access to the cell via a carrier-mediated process and that the discrete carrier is the gene product of OPT1. If enkephalins were entering by a nonspecific mechanism such as simple diffusion or endocytosis, then all strains, not just those expressingOPT1, should be able to utilize this substrate.Several enkephalin antagonists were assayed in this study for their effect on enkephalin transport across Opt1p. DADLE and DPDPE are enzymatically stable delta opioid receptor antagonists that are pentapeptide mimetics. Previous reports indicated that DPDPE gained access to the brain by a saturable, carrier-mediated mechanism in the blood-brain barrier, which has yet to be defined (22.Thomas S.A. Abbruscato T.J. Hruby V.J. Davis T.P. J. Pharmacol. Exp. Ther. 1997; 280: 1235-1240PubMed Google Scholar, 23.Williams S.A. Abbruscato T.J. Hruby V.J. Davis T.P. J. Neurochem. 1996; 66: 1289-1299Crossref PubMed Scopus (74) Google Scholar). Interestingly, transport of DPDPE was not inhibited by Leu-enkephalin in those studies, suggesting either the existence of separate transport systems or a common system with different affinities for these two substrates. A recent report suggests that DPDPE crosses the blood-brain barrier by a phenylarsine oxide-sensitive pathway, suggesting a role for a saturable endocytic mechanism in the in vitro andin situ models studied (24.Egleton R.D. Davis T.P. J. Pharm. Sci. 1999; 88: 392-397Abstract Full Text PDF PubMed Scopus (31) Google Scholar). In the present study, DPDPE and DADLE were weak competitors for Leu-enkephalin transport, indicating that Opt1p interacts with the stable antagonists with differential affinities compared with authentic Leu-enkephalin.Naloxone and naltrexone are synthetic opioid receptor antagonists classically used to reverse the effects of opiate overdose (18.McNicholas L.F. Martin W.R. Drugs. 1984; 27: 81-93Crossref PubMed Scopus (49) Google Scholar). Naltrexone is also used clinically in the treatment of alcoholism. Despite the fact that these compounds are similar in structure to morphine, rather than resembling a peptide, they were effective competitors for Leu-enkephalin transport. The effect appears to be specific for the Opt1p transporter because the presence of the morphine analogs did not influence the activity of the unrelated di- and tripeptide transporter Ptr2p. The nature of the inhibition of Leu-enkephalin transport by naloxone and naltrexone is currently under investigation. Specifically, it would be of interest to determine whether these compounds are substrates for transport or are nonsubstrate competitors for Opt1p.There is increasing evidence that opioids and their analogues enter the central nervous system by carrier-mediated transport across the blood-brain barrier (6.Banks W.A. Kastin A.J. Am. J. Physiol. 1990; 259: E1-E10PubMed Google Scholar, 22.Thomas S.A. Abbruscato T.J. Hruby V.J. Davis T.P. J. Pharmacol. Exp. Ther. 1997; 280: 1235-1240PubMed Google Scholar, 25.Fiori A. Cardelli P. Negri L. Savi M.R. Strom R. Erspamer V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9469-9474Crossref PubMed Scopus (46) Google Scholar). Evidence also exists to suggest that the clearance of the enkephalin analogue DPDPE occurs by saturable efflux from the brain and systemic elimination of intact DPDPE via biliary excretion (26.Chen C. Pollack G.M. J. Pharmacol. Exp. Ther. 1997; 283: 1151-1159PubMed Google Scholar). Furthermore, it is possible that neuronal re-uptake systems exist for enkephalin similar to the well studied transport systems for neurotransmitters such as serotonin and γ-aminobutyric acid (27.Blakely R.D. Ramamoorthy S. Schroeter S. Qian Y. Apparsundaram S. Galli A. DeFelice L.J. Biol. Psychiatry. 1998; 44: 169-178Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 28.Borden L.A. Neurochem. Int. 1996; 29: 335-356Crossref PubMed Scopus (518) Google Scholar). To date, none of the putative transporters for enkephalin have been cloned or characterized at a molecular level. In this report, we present the first evidence for a genetically defined eukaryotic transport protein, Opt1p, which recognizes and translocates both Met- and Leu-enkephalin into an intact eukaryotic cell. The identification of this transporter inSaccharomyces may facilitate the discovery of mammalian homologues, thus providing greater insight into the process of pain and its mediation. Small peptides containing four to five amino acid residues are transported by a recently identified class of peptide transporters named the OPT1 family (1.Lubkowitz M.A. Hauser L. Breslav M. Naider F. Becker J.M. Microbiology. 1997; 143: 387-396Crossref PubMed Scopus (79) Google Scholar, 2.Lubkowitz M.A. Barnes D. Breslav M. Burchfield A. Naider F. Becker J.M. Mol. Microbiol. 1998; 28: 729-741Crossref PubMed Scopus (71) Google Scholar). The amino acid sequence of this family is distinct from that of the PTR family, a ubiquitous group of proton-coupled transporters which selectively transports di- and tripeptides (3.Steiner H.-Y. Naider F. Becker J.M. Mol. Microbiol. 1995; 16: 825-834Crossref PubMed Scopus (202) Google Scholar, 4.Fei Y.J. Ganapathy V. Leibach F.H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 239-261Crossref PubMed Scopus (77) Google Scholar). Phylogenetic analysis suggests that the OPT family is also distinct from the major facilitator superfamily (MFS), a diverse collection of proteins which catalyzes the transport of a wide variety of substrates, including sugars, amino acids, neurotransmitters, and drugs (5.Saier M.H. Adv. Microb. Physiol. 1998; 40: 81-136Crossref PubMed Google Scholar). Members of the OPT family have been identified and characterized in the yeasts Candida albicans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae. Additional members exist in plants, as indicated by searches of publicly accessible data bases. In mammalian tissues, reports in the literature suggest that the enkephalins, endogenous pentapeptides involved in analgesia in the central nervous system, are transported across the blood-brain barrier by a specific, saturable transport system (6.Banks W.A. Kastin A.J. Am. J. Physiol. 1990; 259: E1-E10PubMed Google Scholar). The existence of enkephalin transporters has been inferred from data obtained by measuring whole brain flux of the peptides in rodents (7.Banks W.A. Kastin A.J. Alcohol. 1997; 14: 237-245Crossref PubMed Scopus (24) Google Scholar, 8.Plotkin S.R. Banks W.A. Waguespack P.J. Kastin A.J J. Neurosci. Res. 1997; 48: 273-280Crossref PubMed Scopus (14) Google Scholar, 9.Slokovic B.V. Mackic J.B. Djuricic B.M. Davson H. J. Neurochem. 1989; 53: 1333-1340Crossref PubMed Scopus (81) Google Scholar, 10.Egleton R.D. Abruscato T.J. Thomas S.A. Davis T.P. J. Pharm. Sci. 1998; 87: 1433-1439Abstract Full Text PDF PubMed Scopus (70) Google Scholar). To date, no protein has been identified in eukaryotes as the discrete enkephalin carrier. In this paper, we report that the endogenous opioids Met-enkephalin and Leu-enkephalin, pentapeptides of amino acid sequence YGGFM and YGGFL, respectively, can be transported by cells expressing the S. cerevisiae ORF YJL212C. When expressed under the control of a constitutive promoter in a high copy number vector, this OPT family member is necessary and sufficient to transport Leu-enkephalin into yeast cells. This is the first example of a genetically defined eukaryotic transport protein which can transport enkephalins across the cell membrane. In accordance with the standard nomenclature forS. cerevisiae, we propose the name OPT1 for this gene. DISCUSSIONIn this paper we assign a function to the previously unknown open reading frame YJL212C in the yeast S. cerevisiae and have named this gene OPT1. The protein encoded by OPT1consists of 799 amino acids, and based on the amino acid sequence the predicted protein structure suggests an integral membrane protein containing 12–14 putative membrane-spanning domains. In addition, the protein contains several motifs unique to the OPT family, the largest of which consists of 10 invariable residues (SPYXEVRXXVXXXDDP) located before the first hydrophobic domain (2.Lubkowitz M.A. Barnes D. Breslav M. Burchfield A. Naider F. Becker J.M. Mol. Microbiol. 1998; 28: 729-741Crossref PubMed Scopus (71) Google Scholar). In this study we have confirmed thatOPT1, like other members of the OPT family, encodes a functional oligopeptide transporter.Because Opt1p exhibited all the molecular characteristics of an OPT family member, it was hypothesized that this protein was an oligopeptide transporter, even though it was known that S. cerevisiae could not utilize any tetra- or pentapeptides tested to date to satisfy auxotrophic requirements under routine growth conditions (1.Lubkowitz M.A. Hauser L. Breslav M. Naider F. Becker J.M. Microbiology. 1997; 143: 387-396Crossref PubMed Scopus (79) Google Scholar, 19.Becker J.M. Naider F. Taylor M.D. Amidon G.L. Peptide-based Drug Design: Controlling Transport and Metabolism. American Chemical Society, Washington D. C.1995: 369-384Google Scholar). To see activity of Opt1, it was necessary to express OPT1 under the control of the ADH promoter, a strong, constitutive promoter which would presumably result in high expression of the gene product. In prior studies, Northern blot analysis confirmed that OPT1 was not expressed at detectable levels under routine conditions of logarithmic growth (2.Lubkowitz M.A. Barnes D. Breslav M. Burchfield A. Naider F. Becker J.M. Mol. Microbiol. 1998; 28: 729-741Crossref PubMed Scopus (71) Google Scholar). These results were independently confirmed by serial analysis of gene expression (SAGE) (20.Velculescu V.E. Zhang L. Zhou W. Vogelstein J. Basrai M.A. Bassett Jr., D.E. Heiter P. Vogelstein B. Kinzler K.W. Cell. 1997; 88: 243-251Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar) which revealed that OPT1 is only expressed at a low level (∼1 copy per cell) following nocodazole arrest in the G2/M phase of the cell cycle. Additional analysis of sporulating yeast cells by DNA microarray analysis indicated that OPT1 was expressed during the late stages of sporulation (21.Chu S. DeRisi J. Eisen M. Mulholland J. Botstein D. Brown P.O. Herskowitz I. Science. 1998; 282: 699-705Crossref PubMed Scopus (1510) Google Scholar). In light of these observations, OPT1 gene expression had to be ectopically induced under the control of a heterologous promoter to enable study of Opt1p function in log phase cells.The product of OPT1 is the oligopeptide transporter Opt1p, which translocates pentapeptides, including both Met- and Leu-enkephalin. In BY4730, a strain of S. cerevisiaeauxotrophic for leucine and methionine, only cells expressingOPT1 could grow on Leu-enkephalin in the absence of exogenous leucine. This indicates that enkephalins are transported intact into the cell and then hydrolyzed. If oligopeptides were hydrolyzed by an extracellular protease prior to transport, then the isogenic control strain (BY4730 transformed with the empty vector pDB20), as well as yeast cells transformed with plasmids encoding other OPT family members (CaOPT1, YPR194C) should be able to utilize the hydrolysis products for growth. Chromatographic analysis supports this postulate; no evidence for degraded forms of Leu-enkephalin could be found in the extracellular medium. In addition, a large body of work exists which demonstrates that di- and tripeptides enter the cell intact and are then rapidly hydrolyzed by intracellular peptidases (19.Becker J.M. Naider F. Taylor M.D. Amidon G.L. Peptide-based Drug Design: Controlling Transport and Metabolism. American Chemical Society, Washington D. C.1995: 369-384Google Scholar).Transport of Leu-enkephalin is pH- and temperature-dependent, suggesting that this is a proton-coupled, energy-dependent process. These observations are supported by the sensitivity of the transporter to agents which disrupt the proton gradient or deplete intracellular ATP. Utilization of the transmembrane proton gradient to energize active transport has been demonstrated for the PTR family of di- and tripeptide transporters (4.Fei Y.J. Ganapathy V. Leibach F.H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 239-261Crossref PubMed Scopus (77) Google Scholar). Uptake of radiolabeled Leu-enkephalin was inhibited in the presence of excess unlabeled Met- or Leu-enkephalin; amidated Leu-enkephalin was an ineffective competitor. Tyr-MIF-1 is an amidated tetrapeptide with opiate and anti-opiate activity. This peptide is a substrate for the previously described blood-brain barrier PTS-1 enkephalin transport activity (6.Banks W.A. Kastin A.J. Am. J. Physiol. 1990; 259: E1-E10PubMed Google Scholar) but, like the amidated form of authentic Leu-enkephalin, was not an effective competitor for yeast Opt1p. This observation is consistent with the need for a free carboxyl terminus for substrate recognition by Opt1p. Tetrapeptides were effective inhibitors, with Lys-Leu-Gly-Leu and des-Tyr1 Leu-enkephalin (Gly-Gly-Phe-Leu) eliminating over 50% of radiolabeled enkephalin accumulation, suggesting that an amino-terminal tyrosine is not essential for substrate recognition. Neither the tripeptide enkephalin fragment Gly-Gly-Phe nor the dipeptide Leu-Leu could inhibit uptake, indicating that this system is distinct from Ptr2p and is selective for tetra- and pentapeptides. These data suggest that intact oligopeptides are gaining access to the cell via a carrier-mediated process and that the discrete carrier is the gene product of OPT1. If enkephalins were entering by a nonspecific mechanism such as simple diffusion or endocytosis, then all strains, not just those expressingOPT1, should be able to utilize this substrate.Several enkephalin antagonists were assayed in this study for their effect on enkephalin transport across Opt1p. DADLE and DPDPE are enzymatically stable delta opioid receptor antagonists that are pentapeptide mimetics. Previous reports indicated that DPDPE gained access to the brain by a saturable, carrier-mediated mechanism in the blood-brain barrier, which has yet to be defined (22.Thomas S.A. Abbruscato T.J. Hruby V.J. Davis T.P. J. Pharmacol. Exp. Ther. 1997; 280: 1235-1240PubMed Google Scholar, 23.Williams S.A. Abbruscato T.J. Hruby V.J. Davis T.P. J. Neurochem. 1996; 66: 1289-1299Crossref PubMed Scopus (74) Google Scholar). Interestingly, transport of DPDPE was not inhibited by Leu-enkephalin in those studies, suggesting either the existence of separate transport systems or a common system with different affinities for these two substrates. A recent report suggests that DPDPE crosses the blood-brain barrier by a phenylarsine oxide-sensitive pathway, suggesting a role for a saturable endocytic mechanism in the in vitro andin situ models studied (24.Egleton R.D. Davis T.P. J. Pharm. Sci. 1999; 88: 392-397Abstract Full Text PDF PubMed Scopus (31) Google Scholar). In the present study, DPDPE and DADLE were weak competitors for Leu-enkephalin transport, indicating that Opt1p interacts with the stable antagonists with differential affinities compared with authentic Leu-enkephalin.Naloxone and naltrexone are synthetic opioid receptor antagonists classically used to reverse the effects of opiate overdose (18.McNicholas L.F. Martin W.R. Drugs. 1984; 27: 81-93Crossref PubMed Scopus (49) Google Scholar). Naltrexone is also used clinically in the treatment of alcoholism. Despite the fact that these compounds are similar in structure to morphine, rather than resembling a peptide, they were effective competitors for Leu-enkephalin transport. The effect appears to be specific for the Opt1p transporter because the presence of the morphine analogs did not influence the activity of the unrelated di- and tripeptide transporter Ptr2p. The nature of the inhibition of Leu-enkephalin transport by naloxone and naltrexone is currently under investigation. Specifically, it would be of interest to determine whether these compounds are substrates for transport or are nonsubstrate competitors for Opt1p.There is increasing evidence that opioids and their analogues enter the central nervous system by carrier-mediated transport across the blood-brain barrier (6.Banks W.A. Kastin A.J. Am. J. Physiol. 1990; 259: E1-E10PubMed Google Scholar, 22.Thomas S.A. Abbruscato T.J. Hruby V.J. Davis T.P. J. Pharmacol. Exp. Ther. 1997; 280: 1235-1240PubMed Google Scholar, 25.Fiori A. Cardelli P. Negri L. Savi M.R. Strom R. Erspamer V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9469-9474Crossref PubMed Scopus (46) Google Scholar). Evidence also exists to suggest that the clearance of the enkephalin analogue DPDPE occurs by saturable efflux from the brain and systemic elimination of intact DPDPE via biliary excretion (26.Chen C. Pollack G.M. J. Pharmacol. Exp. Ther. 1997; 283: 1151-1159PubMed Google Scholar). Furthermore, it is possible that neuronal re-uptake systems exist for enkephalin similar to the well studied transport systems for neurotransmitters such as serotonin and γ-aminobutyric acid (27.Blakely R.D. Ramamoorthy S. Schroeter S. Qian Y. Apparsundaram S. Galli A. DeFelice L.J. Biol. Psychiatry. 1998; 44: 169-178Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 28.Borden L.A. Neurochem. Int. 1996; 29: 335-356Crossref PubMed Scopus (518) Google Scholar). To date, none of the putative transporters for enkephalin have been cloned or characterized at a molecular level. In this report, we present the first evidence for a genetically defined eukaryotic transport protein, Opt1p, which recognizes and translocates both Met- and Leu-enkephalin into an intact eukaryotic cell. The identification of this transporter inSaccharomyces may facilitate the discovery of mammalian homologues, thus providing greater insight into the process of pain and its mediation. In this paper we assign a function to the previously unknown open reading frame YJL212C in the yeast S. cerevisiae and have named this gene OPT1. The protein encoded by OPT1consists of 799 amino acids, and based on the amino acid sequence the predicted protein structure suggests an integral membrane protein containing 12–14 putative membrane-spanning domains. In addition, the protein contains several motifs unique to the OPT family, the largest of which consists of 10 invariable residues (SPYXEVRXXVXXXDDP) located before the first hydrophobic domain (2.Lubkowitz M.A. Barnes D. Breslav M. Burchfield A. Naider F. Becker J.M. Mol. Microbiol. 1998; 28: 729-741Crossref PubMed Scopus (71) Google Scholar). In this study we have confirmed thatOPT1, like other members of the OPT family, encodes a functional oligopeptide transporter. Because Opt1p exhibited all the molecular characteristics of an OPT family member, it was hypothesized that this protein was an oligopeptide transporter, even though it was known that S. cerevisiae could not utilize any tetra- or pentapeptides tested to date to satisfy auxotrophic requirements under routine growth conditions (1.Lubkowitz M.A. Hauser L. Breslav M. Naider F. Becker J.M. Microbiology. 1997; 143: 387-396Crossref PubMed Scopus (79) Google Scholar, 19.Becker J.M. Naider F. Taylor M.D. Amidon G.L. Peptide-based Drug Design: Controlling Transport and Metabolism. American Chemical Society, Washington D. C.1995: 369-384Google Scholar). To see activity of Opt1, it was necessary to express OPT1 under the control of the ADH promoter, a strong, constitutive promoter which would presumably result in high expression of the gene product. In prior studies, Northern blot analysis confirmed that OPT1 was not expressed at detectable levels under routine conditions of logarithmic growth (2.Lubkowitz M.A. Barnes D. Breslav M. Burchfield A. Naider F. Becker J.M. Mol. Microbiol. 1998; 28: 729-741Crossref PubMed Scopus (71) Google Scholar). These results were independently confirmed by serial analysis of gene expression (SAGE) (20.Velculescu V.E. Zhang L. Zhou W. Vogelstein J. Basrai M.A. Bassett Jr., D.E. Heiter P. Vogelstein B. Kinzler K.W. Cell. 1997; 88: 243-251Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar) which revealed that OPT1 is only expressed at a low level (∼1 copy per cell) following nocodazole arrest in the G2/M phase of the cell cycle. Additional analysis of sporulating yeast cells by DNA microarray analysis indicated that OPT1 was expressed during the late stages of sporulation (21.Chu S. DeRisi J. Eisen M. Mulholland J. Botstein D. Brown P.O. Herskowitz I. Science. 1998; 282: 699-705Crossref PubMed Scopus (1510) Google Scholar). In light of these observations, OPT1 gene expression had to be ectopically induced under the control of a heterologous promoter to enable study of Opt1p function in log phase cells. The product of OPT1 is the oligopeptide transporter Opt1p, which translocates pentapeptides, including both Met- and Leu-enkephalin. In BY4730, a strain of S. cerevisiaeauxotrophic for leucine and methionine, only cells expressingOPT1 could grow on Leu-enkephalin in the absence of exogenous leucine. This indicates that enkephalins are transported intact into the cell and then hydrolyzed. If oligopeptides were hydrolyzed by an extracellular protease prior to transport, then the isogenic control strain (BY4730 transformed with the empty vector pDB20), as well as yeast cells transformed with plasmids encoding other OPT family members (CaOPT1, YPR194C) should be able to utilize the hydrolysis products for growth. Chromatographic analysis supports this postulate; no evidence for degraded forms of Leu-enkephalin could be found in the extracellular medium. In addition, a large body of work exists which demonstrates that di- and tripeptides enter the cell intact and are then rapidly hydrolyzed by intracellular peptidases (19.Becker J.M. Naider F. Taylor M.D. Amidon G.L. Peptide-based Drug Design: Controlling Transport and Metabolism. American Chemical Society, Washington D. C.1995: 369-384Google Scholar). Transport of Leu-enkephalin is pH- and temperature-dependent, suggesting that this is a proton-coupled, energy-dependent process. These observations are supported by the sensitivity of the transporter to agents which disrupt the proton gradient or deplete intracellular ATP. Utilization of the transmembrane proton gradient to energize active transport has been demonstrated for the PTR family of di- and tripeptide transporters (4.Fei Y.J. Ganapathy V. Leibach F.H. Prog. Nucleic Acids Res. Mol. Biol. 1998; 58: 239-261Crossref PubMed Scopus (77) Google Scholar). Uptake of radiolabeled Leu-enkephalin was inhibited in the presence of excess unlabeled Met- or Leu-enkephalin; amidated Leu-enkephalin was an ineffective competitor. Tyr-MIF-1 is an amidated tetrapeptide with opiate and anti-opiate activity. This peptide is a substrate for the previously described blood-brain barrier PTS-1 enkephalin transport activity (6.Banks W.A. Kastin A.J. Am. J. Physiol. 1990; 259: E1-E10PubMed Google Scholar) but, like the amidated form of authentic Leu-enkephalin, was not an effective competitor for yeast Opt1p. This observation is consistent with the need for a free carboxyl terminus for substrate recognition by Opt1p. Tetrapeptides were effective inhibitors, with Lys-Leu-Gly-Leu and des-Tyr1 Leu-enkephalin (Gly-Gly-Phe-Leu) eliminating over 50% of radiolabeled enkephalin accumulation, suggesting that an amino-terminal tyrosine is not essential for substrate recognition. Neither the tripeptide enkephalin fragment Gly-Gly-Phe nor the dipeptide Leu-Leu could inhibit uptake, indicating that this system is distinct from Ptr2p and is selective for tetra- and pentapeptides. These data suggest that intact oligopeptides are gaining access to the cell via a carrier-mediated process and that the discrete carrier is the gene product of OPT1. If enkephalins were entering by a nonspecific mechanism such as simple diffusion or endocytosis, then all strains, not just those expressingOPT1, should be able to utilize this substrate. Several enkephalin antagonists were assayed in this study for their effect on enkephalin transport across Opt1p. DADLE and DPDPE are enzymatically stable delta opioid receptor antagonists that are pentapeptide mimetics. Previous reports indicated that DPDPE gained access to the brain by a saturable, carrier-mediated mechanism in the blood-brain barrier, which has yet to be defined (22.Thomas S.A. Abbruscato T.J. Hruby V.J. Davis T.P. J. Pharmacol. Exp. Ther. 1997; 280: 1235-1240PubMed Google Scholar, 23.Williams S.A. Abbruscato T.J. Hruby V.J. Davis T.P. J. Neurochem. 1996; 66: 1289-1299Crossref PubMed Scopus (74) Google Scholar). Interestingly, transport of DPDPE was not inhibited by Leu-enkephalin in those studies, suggesting either the existence of separate transport systems or a common system with different affinities for these two substrates. A recent report suggests that DPDPE crosses the blood-brain barrier by a phenylarsine oxide-sensitive pathway, suggesting a role for a saturable endocytic mechanism in the in vitro andin situ models studied (24.Egleton R.D. Davis T.P. J. Pharm. Sci. 1999; 88: 392-397Abstract Full Text PDF PubMed Scopus (31) Google Scholar). In the present study, DPDPE and DADLE were weak competitors for Leu-enkephalin transport, indicating that Opt1p interacts with the stable antagonists with differential affinities compared with authentic Leu-enkephalin. Naloxone and naltrexone are synthetic opioid receptor antagonists classically used to reverse the effects of opiate overdose (18.McNicholas L.F. Martin W.R. Drugs. 1984; 27: 81-93Crossref PubMed Scopus (49) Google Scholar). Naltrexone is also used clinically in the treatment of alcoholism. Despite the fact that these compounds are similar in structure to morphine, rather than resembling a peptide, they were effective competitors for Leu-enkephalin transport. The effect appears to be specific for the Opt1p transporter because the presence of the morphine analogs did not influence the activity of the unrelated di- and tripeptide transporter Ptr2p. The nature of the inhibition of Leu-enkephalin transport by naloxone and naltrexone is currently under investigation. Specifically, it would be of interest to determine whether these compounds are substrates for transport or are nonsubstrate competitors for Opt1p. There is increasing evidence that opioids and their analogues enter the central nervous system by carrier-mediated transport across the blood-brain barrier (6.Banks W.A. Kastin A.J. Am. J. Physiol. 1990; 259: E1-E10PubMed Google Scholar, 22.Thomas S.A. Abbruscato T.J. Hruby V.J. Davis T.P. J. Pharmacol. Exp. Ther. 1997; 280: 1235-1240PubMed Google Scholar, 25.Fiori A. Cardelli P. Negri L. Savi M.R. Strom R. Erspamer V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9469-9474Crossref PubMed Scopus (46) Google Scholar). Evidence also exists to suggest that the clearance of the enkephalin analogue DPDPE occurs by saturable efflux from the brain and systemic elimination of intact DPDPE via biliary excretion (26.Chen C. Pollack G.M. J. Pharmacol. Exp. Ther. 1997; 283: 1151-1159PubMed Google Scholar). Furthermore, it is possible that neuronal re-uptake systems exist for enkephalin similar to the well studied transport systems for neurotransmitters such as serotonin and γ-aminobutyric acid (27.Blakely R.D. Ramamoorthy S. Schroeter S. Qian Y. Apparsundaram S. Galli A. DeFelice L.J. Biol. Psychiatry. 1998; 44: 169-178Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 28.Borden L.A. Neurochem. Int. 1996; 29: 335-356Crossref PubMed Scopus (518) Google Scholar). To date, none of the putative transporters for enkephalin have been cloned or characterized at a molecular level. In this report, we present the first evidence for a genetically defined eukaryotic transport protein, Opt1p, which recognizes and translocates both Met- and Leu-enkephalin into an intact eukaryotic cell. The identification of this transporter inSaccharomyces may facilitate the discovery of mammalian homologues, thus providing greater insight into the process of pain and its mediation. We thank Michael Owston for assistance." @default.
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