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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 gene OPT1. 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 microM. 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.

Oligosaccharyltransferase, the enzyme catalyzing the co-translational transfer of oligosaccharide from dolichyl-PP-GlcNAc2Man9Glc3 to -Asn-X-Ser/Thr- sequences in nascent polypeptide chains, was studied in hen oviduct microsomes using the active site-directed photoaffinity probe 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. Several lines of evidence established that the tripeptide probe interacted with a 57-kDa protein of the endoplasmic reticulum that was subsequently glycosylated and converted to a 60-kDa form. The 57-kDa protein, isolated by two-dimensional gel electrophoresis, was used as immunogen to prepare polyclonal antisera. The specificity of the antibody was established on the basis of its ability to 1) recognize the 57-kDa protein by immunoblotting and 2) immunoprecipitate the photolabeled protein. The antibody also recognized photolabeled protein from different tissues and organisms. The 57-kDa protein isolated by immunoprecipitation retained its ability to interact with the photoaffinity probe but was inactive in catalyzing glycosylation of peptides. This result suggests that the 57-kDa protein is the component of oligosaccharyltransferase that recognizes the glycosylation site in polypeptides. These results are discussed in terms of possible models for the structure of oligosaccharyltransferase in the endoplasmic reticulum.

Intact cells of Saccharomyces cerevisiae 139 hydrolyzed amino acid-p-nitroanilide by an activity similar to that of aminopeptidase II, as well-characterized external peptidase in yeast. In contrast, trimethionine, a model peptide used in transport assays, was not hydrolyzed by this aminopeptidase II-like activity, and the peptidase activity toward this substrate was localized in the soluble fraction of the yeast. We conclude that this tripeptide is taken up by S. cerevisiae intact and rapidly hydrolyzed inside the cell.

A peptide transport mutant of a leucine-lysine auxotroph of Saccharomyces cerevisiae (strain Z1-2D) was isolated on the basis of its resistance to L-ethionyl-L-alanine. The mutant, designated Z1-2D Etar, did not utilize di- and tripeptides containing leucine or lysine although it contained peptidases which released the required amino acids from these substrates. S. cerevisiae Z1-2D Etar did not accumulate radioactivity from [14C]glycyl-L-leucine under conditions identical to those in which the parent took up the label from this dipeptide. These results indicate that the mutant lacks the cellular mechanism to transport peptides to the site of the peptidase activity and that di- and tripeptides share a common mode of entry into yeast.

A variety of leucine-containing di- and tripeptides and two lysine-containing dipeptides supported the growth of strain Z1-2D, a leucine, lysine auxotroph of Saccharomyces cerevisiae. However, (Lys)2, (Lys)3, (Lys)4, and (Lys)5 as well as Gly-Leu-Gly, three tetra- and one pentapeptide containing leucine were not utilized by the mutant. Cellular peptidases released leucine or lysine from all of these non-growth-supporting peptides, suggesting that the failure of strain Z1-2D to utilize these compounds reflects their failure to enter the yeast. Competition studies employing phenylalanine or non-leucine-containing peptides showed that the uptake of peptides into S. cerevisiae Z1-2D is distinct from that of amino acids and that di- and oligopeptides may share a common transport system. The failure of strain Z1-2D to utilize any peptide larger than (Leu)3 may indicate a transport size limit. Such a size limit would influence the construction of models that explain the action of yeast mating factors.

At least four distinct aminopeptidase activities and a single dipeptidase activity were found in cell extracts of a leucine-lysine auxotroph of Saccharomyces cerevisiae. The assay for peptidase activity involved polyacrylamide gel electrophoresis followed by an enzyme-coupled activity staining procedure. The aminopeptidases had largely overlapping specificities but could be distinguished from one another by their electrophoretic mobilities and activities toward different peptide substrates. Substrates tested included both free and blocked di- and tripeptides and amino acid derivatives.

A plasmid vector was developed that permitted high-level expression of a functional form of the Saccharomyces cerevisiae alpha-factor receptor (the STE2 gene product) tagged at its C-terminal end with an epitope (FLAG) and a His6 tract. When expressed in yeast from this plasmid, Ste2p was produced at a level at least 3-fold higher than that reported previously for any other 7-transmembrane-segment receptor expressed in the same cells. For purification, isolated cell membranes containing the overexpressed receptor were solubilized with detergent under specific conditions and subjected to immobilized metal affinity chromatography. Yields as high as 1 mg of nearly homogeneous (95%) receptor were routinely obtained even from relatively small scale preparations (60 g of frozen cell paste). The purified receptor was reconstituted into artificial phospholipid vesicles. Radioligand binding studies demonstrated that the purified receptor, in the reconstituted vesicles, bound its tridecapeptide ligand (alpha-factor) with a KD (155 nM) consistent with the affinity expected for this receptor in the absence of its associated G protein. Efficient restoration of ligand binding activity upon reconstitution required the addition of solubilized membranes prepared from a yeast strain lacking the receptor. Sufficient amounts of active material can be obtained by this procedure to allow physical studies of this receptor and other 7-transmembrane-segment receptors expressed in this system.

In a microsome system rendered competent in protein translation by the addition of rabbit reticulocyte lysate, co-translational insertion and glycosylation of N-linked glycoproteins is observed when the appropriate mRNA is supplied. We have utilized this system to examine the ability of acceptor tripeptides of the type Asn-X-Thr/Ser to inhibit co-translational glycosylation. Using endogenous oligosaccharide-lipid as the carbohydrate donor, dog pancreas microsomes efficiently glycosylated N alpha-[3H]Ac-Asn-Leu-Thr-NHCH3 (apparent Km = 100 microM). Glycopeptide formation was essentially complete within 20 min. In the presence of mRNA from vesicular stomatitis virus or chicken ovalbumin, a similar tripeptide, N alpha-Ac-Asn-Leu-Thr-NH2, inhibited co-translational glycosylation. Translocation of the nascent chains was not affected. Thus, in the absence of peptide, all translated G protein was glycosylated and found within the microsomes, whereas in the presence of the peptide a mixture of glycosylated and nonglycosylated G protein was sequestered. Inhibition of nascent chain glycosylation was competitive and not merely the result of oligosaccharide lipid depletion, because preincubation of the microsomes with the peptide followed by its removal did not affect subsequent glycosylation of ovalbumin or G protein. Six derivatives of Asn-Leu-Thr-NH2, three of which were acceptors and three of which were not, were tested for their ability to inhibit co-translational glycosylation. The three acceptor peptides, N alpha-Ac-Asn-Leu-Thr-NH2, N alpha-Oc-Asn-Leu-Thr-NH2, and N alpha-Bz-Asn-Leu-Thr-NH2, effectively inhibited nascent chain glycosylation. In contrast, the three nonacceptors, N alpha-Ac-Gln-Leu-Thr-NH2, N alpha-Ac-Asn(N beta-Me)-Leu-Thr-NH2, and Asn-Leu-Thr-NH2, had no effect. Taken together, these data indicate that the inhibition of co-translational glycosylation by a peptide is dependent on its ability to compete for the active site of the oligosaccharyl transferase.

The minimum primary structural requirement for N-glycosylation of proteins is the sequence -Asn-X-Thr/Ser-. In the present study, NH2-terminal derivatives of Asn-Leu-Thr-NH2 and peptides with asparagine replacements have been tested as substrates or inhibitors of N-glycosylation. The glycosylation of a known acceptor, N alpha-[3H]Ac-Asn-Leu-Thr-NHCH3, was optimized in chicken oviduct microsomes. The reaction was shown to be dependent upon Mn2+ and linear for 10 min at 30 degrees C; the apparent Km for the peptide was found to be 10 microM. N alpha-Acyl derivatives of Asn-Leu-Thr-NH2 (N-acetyl, N-benzoyl, N-octanoyl, or N-t-butoxycarbonyl) inhibited the glycosylation of N alpha-[3H] Ac-Asn-Leu-Thr-NHCH3 in a dose-dependent manner; additional experiments demonstrated that these compounds were alternative substrates rather than true inhibitors. The benzoyl and octanoyl derivatives were 10 times as effective as N alpha-Ac-Asn-Leu-Thr-NH2 in inhibiting glycosylation. In contrast, peptides containing asparagine modifications or substitutions were neither substrates nor inhibitors of N-glycosylation. They did not compete for glycosylation of 3H-peptide at 100-fold greater concentrations, and did not deplete endogenous pools of oligosaccharide-lipid. Thus, the asparagine side chain is an absolute requirement for recognition by the transferase. The majority of the glycosylated product (61%), but only 1% of the unglycosylated peptide, remained associated with the microsomes after high speed centrifugation. A large 41-amino acid residue acceptor peptide, alpha-lac17-58, was a poor substitute for glycosylation unless detergent was added to the microsomes. In contrast, glycosylation of tripeptide acceptors was not stimulated by detergent. Both of these findings suggest that the tripeptides are freely permeable to the microsomal membrane and support the earlier conclusion that glycosylation of proteins occurs at the luminal face of the microsomes.

Oligosaccharyltransferase, the enzyme that catalyzes the transfer of the oligosaccharide chain of dolichol-P-P-GlcNAc2Man9Glc3 to asparagine residues in -Asn-X-Thr/Ser- sites within polypeptides, has been radiolabeled using a photoactivatable azido tripeptide acceptor, N alpha-[3H]Ac-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the oligosaccharyltransferase polypeptide from hen oviduct microsomes is 60 kDa. Radiolabeling of the 60-kDa polypeptide was completely dependent upon photolysis of hen oviduct endoplasmic reticulum preparations in the presence of the azido peptide and Mn2+, which is required for enzymatic activity. Labeling of the enzyme was not inhibited in the presence of a 10-fold excess of the nonacceptor peptides, unacetylated Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2 or unacetylated Asn-Leu-Thr-NH2, whereas it was completely abolished by the presence of a 10-fold excess of the competing acceptor peptide, N alpha-Bz-Asn-Leu-Thr-NH2. Thermal inactivation of oligosaccharyltransferase was achieved by heating endoplasmic reticulum preparations to 60 degrees C. This loss of enzyme activity at 60 degrees C paralleled a comparable decrease in radiolabeling of the 60-kDa polypeptide, whereas temperatures of 50 degrees C and lower had no effect on either process. Oligosaccharyltransferase itself may be an N-linked glycoprotein, because the 60-kDa radiolabeled polypeptide binds to concanavalin A-agarose and is susceptible to digestion by beta-endohexosaminidase H.

alpha-Factor, a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae. An analogue of alpha-factor, [DHP8,DHP11,Nle12] tridecapeptide (where DHP represents 3,4-dehydro-L-proline and Nle represents norleucine), was catalytically reduced in the presence of 3H gas to produce a radiolabeled pheromone with high specific activity, purity, and biological activity. Association and dissociation kinetics indicated values of 4.9 x 10(4) M-1 s-1 for k1 and 1.1 x 10(-3) s-1 for k-1. Saturation binding studies gave an equilibrium dissociation constant equal to 2.3 x 10(-8) M, which approximated the kinetically derived KD of 2.2 x 10(-8) M. These values compare favorably to the previously determined KD of 6 x 10(-9) M (Jenness, D.D., Burkholder, A.C., and Hartwell, L.H. (1986) Mol. Cell. Biol. 6, 318-320). Scatchard analysis and dissociation in the presence of excess unlabeled ligand indicated interaction with a homogeneous population of noninteracting binding sites (13,000 sites/cell). A number of alpha-factor analogues, previously investigated for their structure-function relationships (Naider, F., and Becker, J.M. (1986) CRC Crit. Rev. Biochem. 21, 225-249), were used to compete with [3H]alpha-factor binding. Four tridecapeptides having conservative amino acid replacements bound strongly to the receptor. In contrast, [Phe3]alpha-factor and 10 des-Trp1-alpha-factor analogues bound to the receptor 1-3 orders of magnitude less effectively than did alpha-factor itself. The binding constants for all active pheromones correlated with biological activity. However, des-Trp1[Phe3]alpha-factor and des-Trp1-[Ala3]alpha-factor, which were not biologically active, still competed with alpha-factor binding, indicating that these analogues fail to induce a secondary signal necessary for biological response to the pheromone. One analogue, des-Trp1-[Cha3,L-Ala9]alpha-factor (where Cha represents cyclohexylalanine), was not biologically active and did not demonstrate binding to the receptor, whereas des-Trp1-[Cha3,D-Ala9]alpha-factor was active and bound to the receptor. This finding suggests that a type II beta-turn is necessary for binding of alpha-factor to its receptor and for subsequent biological activity.

Cysteine-containing amino acid sequences (CAAX, CC, and CXC; C is cysteine, A is any aliphatic amino acid, and X is any amino acid) are targets for the attachment of C15 (farnesyl) and C20 (geranylgeranyl) isoprenoids to peptides and proteins by specific prenyltransferases. Although much work has centered on the enzymatic mechanisms of these enzymes, the biological consequences of the differential isoprenylation they catalyze remain to be elucidated. Farnesylation of the a-factor mating pheromone of Saccharomyces cerevisiae is a known prerequisite for its biological activity and its secretion through a pathway utilizing the yeast STE6 protein, a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein. We generated specific mutations in the a-factor gene to encode isoprenylation targets for geranylgeranylation [Cys-Val-Ile-Leu (CVIL) and Ser-Val-Cys-Cys (SVCC)] in place of the natural farnesylation motif [Cys-Val-Ile-Ala (CVIA)]. The a-factors containing these modified prenylation sites were successfully exported by a STE6-dependent mechanism. Furthermore, these peptides, as well as synthetic geranylgeranyl a-factor, retained bioactivity. Chromatographic comparisons of synthetic and biosynthetic pheromones suggest that, in vivo, a peptide substrate containing the geranylgeranylation target CVIL can be both farnesylated and geranylgeranylated. These results clearly demonstrate that in vivo (i) different prenyltransferases may recognize the same substrate; (ii) both farnesylated and geranylgeranylated a-factor peptides are substrates for export via STE6, a MDR-like protein; and (iii) farnesylated and geranylgeranylated pheromones are both biologically active.

Prototrophic Saccharomyces cerevisiae X2180, when grown on unsupplemented minimal medium, displayed little sensitivity to ethionine- and m-fluorophenylalanine-containing toxic dipeptides. We examined the influence of the 20 naturally occurring amino acids on sensitivity to toxic dipeptides. A number of these amino acids, at concentrations as low as 1 microM (leucine and tryptophan), produced large increases in sensitivity to leucyl-ethionine, alanyl-ethionine, and leucyl-m-fluorophenylalanine. Sensitivity to ethionine and m-fluorophenylalanine remained high under either set of conditions. The addition of 0.15 mM tryptophan to a growing culture resulted in the induction of dipeptide transport, as indicated by a 25-fold increase in the initial rate of L-leucyl-L-[3H]leucine accumulation. This increase, which was prevented by the addition of cycloheximide, began within 30 min and peaked approximately 240 min after a shift to medium containing tryptophan. Comparable increases in peptidase activity were not apparent in crude cell extracts from tryptophan-induced cultures. We concluded that S. cerevisiae possesses a specific mechanism for the induction of dipeptide transport that can respond to very low concentrations of amino acids.

An analog of α-factor, the Saccharomyces cerevisiaetridecapeptide mating pheromone (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr), in which the side chains of Lys7and Gln10were covalently linked, was synthesized using solid phase methodologies. The yield of the purified cyclic analog cyclo7,10[Nle12]α-factor was 30%, and its structure was verified by amino acid analysis, peptide sequencing, fast atom bombardment-mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Cyclo7,10[Nle12]α-factor caused growth arrest and morphological alterations in S. cerevisiaeMATa cells qualitatively identical to those induced by linear pheromone and was one-fourth to one-twentieth as active as the linear α-factor depending upon the S. cerevisiaestrain tested. Consistent with the relative activities of the linear and cyclic peptides, binding competition studies indicated that cyclo7,10[Nle12]α-factor had approximately 20–40-fold less affinity for the α-factor receptor. Hydrolysis of the cyclic peptide by the target cells did not lead to opening of the ring and was less rapid than that of linear α-factor. The α-factor antagonist des-Trp1-[Ala3,Nle12]α-factor reversed the activity of the cyclic analog, and cyclo7,10[Nle12]α-factor was not active at the restrictive temperature in a temperature-sensitive receptor mutant. These results support the conclusion that the cyclic α-factor occupies the same binding site within the receptor as is occupied by the natural pheromone. The cyclic α-factor represents a rare example of an agonist among covalently constrained congeners of small linear peptide messengers.

The precursor predicted by the nucleotide sequence of the MF alpha 2 gene of Saccharomyces cerevisiae contains one copy of the tridecapeptide alpha-factor previously characterized (H2N-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-COOH) and one copy of a peptide that contains two conservative amino acid substitutions (H2N-Trp-His-Trp-Leu-Asn-Leu-Arg-Pro-Gly-Gln-Pro-Met-Tyr-COOH). To determine whether the novel molecule possesses biological activity, the Asn-5,Arg-7 tridecapeptide was prepared chemically by solid-phase peptide synthesis. Growth arrest and morphogenesis assays gave identical activity profiles for the Asn-5,Arg-7 peptide and the other gene product, the Gln-5,Lys-7 peptide. The activities of the two peptides were additive and indistinguishable for S. cerevisiae X2180-1A. When present in fourfold molar excess, the biologically inactive desTrp-1,Ala-3 dodecapeptide reversed activity of the Asn-5,Arg-7 and Gln-5,Lys-7 tridecapeptides. Furthermore, neither peptide caused growth arrest of a MATa ste2(Ts) mutant when assayed at the restrictive temperature. These studies suggest that both pheromones interact with the alpha-factor receptor in a similar manner.

Eight synthetic analogs of the mating pheromone alpha-factor-induced morphogenesis and increased agglutinability in a cells. Most analogs induced increased agglutinability at lower concentrations than those at which they induced morphogenesis, but the ratio of the potencies for the two effects varied 140-fold among different analogs. Morphological response to pheromone required exposure for at least 90 min, but increased agglutinability followed exposures of 20 s. Two synthetic analogs induced neither response. In competition experiments, both of these analogs prevented induction of increased agglutinability and morphogenesis by active alpha factor. The inactive peptides blocked increased agglutinability at lower concentrations than those at which they blocked morphogenesis. alpha factors exhibited different structure-function relationships for morphogenesis as compared with agglutinability. Thus, response of Saccharomyces cerevisiae to alpha factor is complex and may be mediated by more than one receptor.

The ability of Escherichia coli K-12 4212 to utilize a variety of oligopeptides as sources of required amino acids was examined. Triornithine-resistant mutants of this strain were oligopeptide permease deficient (Opp-) as judged by their inability to utilize (Lys)3 and (Lys)4 as sources of lysine and their resistance to the toxic tripeptide (Val)3. These same mutants were able to grow when Met-Met-Met, Met-Gly-Met, Met-Gly-Gly, Gly-Met-Gly, Gly-Gly-Met, Gly-Met-Met, Met-Met-Gly, or Leu-Leu-Leu were supplied in place of the requisite amino acid. The system mediating the uptake of these peptides, herein designated Opr I, was not able to transport N-alpha-acetylated peptides, nor the tetrapeptides Met-Gly-Met-Met, Met-Met-Gly-Met, or Met-Met-Met-Gly. Competition experiments indicated that trimethionine and trileucine enter E. coli K-12 via either Opp or Opr I. Analogous results were found using the methionine, leucine-requiring auxotroph E. coli B163. It appears that more than one oligopeptide transport system exists in E. coli and that the system mediating peptide uptake is complex.

Two novel polyoxins, N-epsilon-(octanoyl)-lysyl-uracil polyoxin C (Oct-Lys-UPOC) and N-gamma-(octyl)-glutaminyluracil polyoxin C (Oct-Gln-UPOC), were synthesized by reacting uracil polyoxin C with the appropriate amino acid p-nitrophenyl ester. Oct-Lys-UPOC and Oct-Gln-UPOC were strong inhibitors (Kis = 1.7 X 10(-6)M) of chitin synthetase from Candida albicans membrane preparations. In a permeabilized-cell assay, Oct-Gln-UPOC had a 10-fold-lower inhibitory activity toward chitin synthetase than did the Oct-Lys-UPOC analog. Both compounds were resistant to hydrolysis by a cell extract of C. albicans H317; however, Oct-Gln-UPOC was hydrolyzed with a half-life of 23 min by a permeabilized-cell preparation. Oct-Lys-UPOC was resistant to hydrolysis by permeabilized cells. Oct-Gln-UPOC and Oct-Lys-UPOC did not compete with the transport of peptides or uridine into the cell. At concentrations up to 2 mM these two new polyoxins were ineffective in the inhibition of cell growth or reduction of cell viability, but they induced aberrant morphologies in C. albicans at a concentration of 0.25 mM. These data suggest that polyoxins containing hydrophobic amino acids retain strong chitin synthetase inhibitory activity and are resistant to cellular hydrolysis. They provide the first example of effective synthetic chitin synthetase inhibitors which are stable inside C. albicans.

Three tripeptidyl polyoxins were synthesized and found to inhibit Candida albicans. Compared with the naturally occurring polyoxin D, the three synthetic polyoxins had little effect on chitin synthetase when assayed with a C. albicans membrane preparation. However, all the compounds inhibited growth, affected cell morphology in a manner similar to that of polyoxin D, and were hydrolyzed by cell extracts of C. albicans. Hydrolysis did not occur extracellularly, and at least one of the synthetic polyoxins, leucyl-norleucyl-uracil polyoxin C, inhibited peptide uptake, suggesting entrance into the cell via the peptide transport system. Thus, the intact tripeptidyl polyoxins are inactive prodrugs that are converted to active moieties by cellular enzymes.

We demonstrated that polyoxin D at millimolar concentrations caused marked morphological alterations of the human pathogens Candida albicans and Cryptococcus neoformans. C. albicans incubated in the presence of this drug grew in long chains that were severely swollen. Polyoxin D inhibited the growth of C. neoformans and killed cells of both the yeast and the hyphal phase of C. albicans. These observations give the first evidence that polyoxin antibiotics can kill zoopathogenic fungi.