Transport and hydrolysis of peptides in Saccharomyces cerevisiae

The transport and hydrolysis of several radioactive di- and tripeptides in Saccharomyces cerevisiae was studied. A peptide-transport-deficient mutant isolated on the basis of its resistance to nikkomycin Z lost most of its capacity to take up di- and tripeptides. The transport kinetics of [14C]methionylglycine, [14C]methionylsarcosine and [3H]nikkomycin Z indicated that peptide transport is not dependent on intracellular hydrolysis. Intact cells had some peptidase activity towards methionylsarcosine but not towards nikkomycin Z. The relationship between this activity and peptide transport is discussed.     

Isolation of a peptide transport-deficient mutant of yeast.

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.     

Toxicity of oxalysine and oxalysine-containing peptides against Candida albicans: regulation of peptide transport by amino acids.

A lysine antimetabolite, L-4-oxalysine [H2NCH2CH2OCH2CH(NH2)COOH], and oxalysine-containing di-, tri-, tetra- and pentapeptides inhibited growth of Candida albicans H317. Micromolar amounts of amino acids were found to overcome ammonium repression of the di- and tripeptide transport system(s) in strain H317. Several amino acids increased the toxicity of oxalysine-containing di- and tripeptides for C. albicans with little or no increase in toxicity of oxalysine or oxalysine-containing tetra- and pentapeptides. L-Lysine completely reversed the toxicity of oxalysine by competing with the transport of oxalysine into the cells. In contrast, L-lysine increased the toxicity of oxalysine-containing di- and tripeptides, but had no effect on the toxicity of oxalysine-containing tetra- and pentapeptides. Incubation of cells with L-lysine for 4 h resulted in a 15-fold increase in the rate of transport of radiolabelled dileucine, indicating that increased sensitivity of C. albicans to some toxic peptides in the presence of L-lysine may be attributed to an increased rate of transport of these peptides. Our results indicate that the dipeptide and tripeptide transport system(s) of C. albicans are regulated by micromolar amounts of amino acids in a similar fashion to the regulation of peptide transport in Saccharomyces cerevisiae and that multiple peptide transport systems differentially regulated by various nitrogen sources and amino acids exist in C. albicans.     

Peptide uptake is essential for growth of Lactococcus lactis on the milk protein casein.

The chlorated dipeptide L-alanyl-beta-chloro-L-alanine (diACA) is very toxic for Lactococcus lactis. Spontaneous mutants resistant to the dipeptide were isolated from plates. The presence and activities of cell wall-associated proteinase, different peptidases in cell extracts, amino acid transport systems, and di- and oligopeptide transport systems were examined and compared in a diACA-resistant mutant and the wild type. Only the rates of di- and tripeptide transport were found to be significantly reduced in the diACA-resistant mutant of L. lactis ML3. Since all other characteristics of this mutant were comparable to those of the wild type, the diACA-resistant mutant is most likely deficient in di- and tripeptide transport. Uptake of di- and tripeptides by L. lactis ML3 was found to be mainly mediated by one peptide transport system. The peptide transport-deficient mutant was found to be unable to grow on a chemically defined medium supplemented with casein as the sole nitrogen source, whereas growth could be restored by the addition of amino acids. These results indicate that peptide transport in L. lactis ML3 is an essential component in the process of casein utilization during growth in milk.     

Substrate specificity of peptide permeases in Candida albicans

Abstract Specificity of peptide transport systems in Candida albicans was studied using as an experimental tool novel anticandidal peptides, containing the N³-4-methoxyfumaroyl- l -2,3-diamino-propanoic acid residue. Studies on cross-resistance and on peptide uptake by spontaneous mutants resistant to toxic peptides, confirmed the multiplicity of peptide permeases in Candida albicans . At least two peptide permeases exist in this microorganism; the first one, specific for di- and tripeptides and the second, for oligopeptides containing 3–6 amino acids. The rate of the tritetra tetra-, penta- and hexapeptide transport in the mycelial form of Candida albicans is about 2-times higher than in the yeast form, while that of dipeptides is markedly reduced.
Tripeptides are proposed as the most efficient carriers for the delivery of 'warhead' amino acids into Candida albicans cells.     

Mechanism of action of nikkomycin and the peptide transport system of Candida albicans

Nikkomycin was found to be a potent growth inhibitor of Candida albicans through competitive inhibition of chitin synthase [Ki = 0.16 microM (0.1 microgram ml-1)]. The activity of the peptide-nucleoside drug was antagonized by both peptone and defined peptides. Transported dipeptides were effective antagonists while transported oligopeptides were not. A mutant of C. albicans resistant to the effects of nikkomycin through a transport defect was unable to transport dipeptides, while oligopeptide uptake was apparently unaffected. At least two peptide permeases are operational in this organism.     

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.     

Peptide transport in yeast: utilization of leucine- and lysine-containing peptides by Saccharomyces cerevisiae.

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.     

Mutation affecting peptide bond formation in nikkomycin biosynthesis

Nikkomycin, a nucleoside-peptide analog of UDP-N-acetylglucosamine, is a potent chitin synthase inhibitor produced by the bacterium Streptomyces tendae. The HPLC profile of fermentation products in culture broths of a non-producing mutant, Nik 15, was compared with nikkomycin standards. Nikkomycin C and D, the glycone and aglycone moieties, respectively, of nikkomycin Z accumulated. This indicates the mutation affects the capacity to form a peptide bond between nikkomycin C and D, which is here proposed to be the terminal step in the synthesis of the biologically active nikkomycin Z. This is also the first documented case of a mutation affecting a specific step in nikkomycin biosynthesis.     

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.     

Di-tripeptides and oligopeptides are taken up via distinct transport mechanisms in Lactococcus lactis.

Lactococcus lactis ML3 possesses two different peptide transport systems of which the substrate size restriction and specificity have been determined. The first system is the earlier-described proton motive force-dependent di-tripeptide carrier (E. J. Smid, A. J. M. Driessen, and W. N. Konings, J. Bacteriol. 171:292-298, 1989). The second system is a metabolic energy-dependent oligopeptide transport system which transports peptides of four to at least six amino acid residues. The involvement of a specific oligopeptide transport system in the utilization of tetra-alanine and penta-alanine was established in a mutant of L. lactis MG1363 that was selected on the basis of resistance to toxic analogs of alanine and alanine-containing di- and tripeptides. This mutant is unable to transport alanine, dialanine, and trialanine but still shows uptake of tetra-alanine and penta-alanine. The oligopeptide transport system has a lower activity than the di-tripeptide transport system. Uptake of oligopeptides occurs in the absence of a proton motive force and is specifically inhibited by vanadate. The oligopeptide transport system is most likely driven by ATP or a related energy-rich, phosphorylated intermediate.     

A di- and tripeptide transport system can supply Listeria monocytogenes Scott A with amino acids essential for growth.

Listeria monocytogenes takes up di- and tripeptides via a proton motive force-dependent carrier protein. This peptide transport system resembles the recently cloned and sequenced secondary di- and tripeptide transport system of Lactococcus lactis (A. Hagting, E. R. S. Kunji, K. J. Leenhouts, B. Poolman, and W. N. Konings, J. Biol. Chem. 269:11391-11399, 1994). The peptide permease of L. monocytogenes has a broad substrate specificity and allows transport of the nonpeptide substrate 5-aminolevulinic acid, the toxic di- and tripeptide analogs, alanyl-beta-chloroalanine and alanyl-alanyl-beta-chloroalanine, and various di- and tripeptides. No extracellular peptide hydrolysis was detected, indicating that peptides are hydrolyzed after being transported into the cell. Indeed, peptidase activities in response to various synthetic substrates were detected in cell extracts obtained from L. monocytogenes cells grown in brain heart infusion broth or defined medium. The di- and tripeptide permease can supply L. monocytogenes with essential amino acids for growth and might contribute to growth of this pathogen in various foods where peptides are supplied by proteolytic activity of other microorganisms present in these foods. Possible roles of this di- and tripeptide transport system in the osmoregulation and virulence of L. monocytogenes are discussed.     

Synergistic action of nikkomycin X/Z with azole antifungals on Candida albicans.

Fluconazole, ketoconazole and tioconazole were shown to act synergistically in vitro with the antibiotic nikkomycin X/Z on the pathogenic fungus Candida albicans. The phenomenon was demonstrated using a checkerboard technique and growth inhibition experiments. The azole antifungal agents, even at concentrations not affecting growth, decreased the incorporation of the 14C-label from [14C]glucose into chitin of the candidal cell wall. After 3 h incubation with tioconazole, 1 microgram ml-1, the incorporation of the radiolabelled glucose into chitin of intact cells and regenerating spheroplasts of C. albicans was inhibited by 43% and 30%, respectively. Moreover, the relative chitin content was approximately 45% lower than that of control cells. The chitin content increased after prolonged incubation with azoles, thus confirming the known phenomenon of azole-induced uncoordinated chitin synthesis and deposition. On the other hand, azole derivatives had very little effect on the rate of nikkomycin transport into C. albicans cells. A sequential blockade mechanism of synergism is proposed.     

Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans

The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was addressed through the characterization of C. albicans strains without a functional HOG1 gene. Analysis of the phenotype of hog1 mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions, hog1 mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the hog1 mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in C. albicans. We show that C. albicans hog1 mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that HOG1 overexpression in Saccharomyces cerevisiae represses the pseudodimorphic transition. Most interestingly, deletion of HOG1 resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy.     

Peptide transport in rabbit kidney. Studies with l-carnosine

l-Carnosine was shown to be transported into rabbit renal brush-border membrane vesicles by an Na⁺ - independent mechanism. The transport was competitively inhibited by glycyl-l-proline. Various di- and tripeptides inhibited l-carnosine transport, whereas free amino acids did not. Inhibition studies showed that blocking the free amino and carboxyl groups of the peptide reduced its affinity for the transport carrier. Under the conditions in which there was no detectable hydrolysis of l-carnosine in the medium, intravesicular contents showed a 30% hydrolysis of the peptide within the vesicles. Disruption of membrane vesicles with deoxycholate resulted in a 3-fold increase in l-carnosine hydrolyzing activity over untreated intact vesicles. Based on these observations, a model for peptide transport is proposed in which transport of the intact peptide across the membrane is followed by its partial or complete hydrolysis by a membrane peptidase whose active site is on the cytoplasmic side of the membrane.     

Histidine aminotransferase activity in Streptomyces tendae and its correlation with nikkomycin production

Streptomyces tendae Tü901 produces nikkomycins belonging to the nucleoside peptide antibiotics. Mutants defective in histidine catabolism were isolated and characterized with regard to their histidine ammonium-lyase activity and antibiotic synthesis. In the histidine ammonialyase-negative mutant hut-11 which was unimpaired in nikkomycin production histidine aminotransferase activity was detected as an additional histidine metabolizing enzyme. A protein exhibiting histidine aminotransferase activity could be demonstrated on non-denaturing gels of hut-11 crude extracts. Using optimized assay conditions, histidine aminotransferase activity was investigated in the strain hut-11 during growth in nikkomycin production medium. Maximal activity was reached at the end of exponential growth prior to nikkomycin production. In the presence of bromopyruvate, an effective inhibitor of histidine aminotransferase activity in vitro, production of nikkomycin Z and X was markedly reduced in hut-11.     

Cloning and functional expression in Escherichia coli of the gene encoding the di- and tripeptide transport protein of Lactobacillus helveticus.

The gene encoding the di- and tripeptide transport protein (DtpT) of Lactobacillus helveticus (DtpTLH) was cloned with the aid of the inverse PCR technique and used to complement the dipeptide transport-deficient and proline-auxotrophic Escherichia coli E1772. Functional expression of the peptide transporter was shown by the uptake of prolyl-[14C] alanine in whole cells and membrane vesicles. Peptide transport via DtpT in membrane vesicles is driven by the proton motive force. The system has specificity for di- and tripeptides but not for amino acids or tetrapeptides. The dtpTLH gene consists of 1,491 bp, which translates into a 497-amino-acid polypeptide. DtpTLH shows 34% identity to the di- and tripeptide transport protein of Lactococcus lactis and is also homologous to various peptide transporters of eukaryotic origin, but the similarity between these proteins is confined mainly to the N-terminal halves.     

Multiplicity of peptide permeases in Candida albicans: evidence from novel chromophoric peptides.

Evidence is presented for the presence of multiple peptide permeases in the eucaryotic organism Candida albicans. Instrumental in these studies were the peptides L-alanyl-L-2-thiophenylglycine (Ala-alpha-TPG) and L-alanyl-L-2-thiophenylglycyl-L-alanine (Ala-alpha-TPG-Ala), which contain thiophenol attached to the alpha-carbon of glycine. Subsequent to transport into the fungal cell, enzymatic hydrolysis of these peptides resulted in the release of free thiophenol, which was quantified by using Ellman reagent. Thiophenol release was shown to be directly correlated to peptide transport and hydrolysis, with transport being the rate-limiting step in intact cells. These peptides, whose uptake showed Michaelis-Menten kinetics, have been used to determine peptide uptake in C. albicans. In addition, we found that the intracellular peptidases can readily be assayed in permeabilized cells and that bestatin, an aminopeptidase inhibitor, inhibits all detectable peptidase activity. C. albicans 124 was able to transport and hydrolyze both Ala-alpha-TPG and Ala-alpha-TPG-Ala, whereas the mutant (124NIK5) was able to transport only the tripeptide. The intracellular peptidases of this mutant were unaffected. In wild-type C. albicans 124, oligopeptides were able to compete with uptake of Ala-alpha-TPG-Ala to a far greater extent than with that of Ala-alpha-TPG; dipeptides inhibited uptake of both Ala-alpha-TPG and Ala-alpha-TPG-Ala. These results provide complementary evidence for the existence of distinct transport systems.     

Conformational limitations of glycylsarcosine as a prototypic substrate for peptide transporters

Peptide transporters are present in all species to absorb the small peptides that occur ubiquitously as products of proteolysis. The broad substrate specificities of these systems allow them to be exploited therapeutically for delivery of peptidomimetic drugs in microbes and man. To this end, glycylsarcosine is currently used as a standard substrate for assaying peptidomimetic transport by peptide transporters. However, in this study we find it is unsuitable as a general substrate, based on assays of its transport by model bacterial peptide transporters and computer-based conformational analysis of its structure. Of the two generic transporters for di- and tripeptides, exemplified by Dpp and Tpp in Escherichia coli, only Dpp can transport glycylsarcosine. The explanation for this finding came from molecular modelling, which indicated that glycylsarcosine can adopt only a restricted range of conformers compared with typical dipeptides, and that of the conformers with a trans peptide bond, the majority have the specific psi and phi backbone torsion angles needed for molecular recognition and transport by Dpp but none possessed psi and phi torsions required for recognition by Tpp; moreover, 38% of its conformers have cis peptide bonds that are not substrates for any peptide transporter. Thus, using glycylsarcosine as substrate in competition assays with compounds that typically form conformers recognised by both types of peptide transporter will underestimate their transport. These findings have implications for assays of oral availability of peptidomimetic drugs such as beta-lactams, ACE inhibitors and anti-viral compounds, for which glycylsarcosine is routinely used.     

Identification and Characterization of <Emphasis Type="Italic">sanH</Emphasis> and <Emphasis Type="Italic">sanI</Emphasis> Involved in the Hydroxylation of Pyridyl Residue During Nikkomycin Biosynthesis in <Emphasis Type="Italic">Streptomyces ansochromogenes</Emphasis>

Nikkomycins are highly potent inhibitors of chitin synthase. The nikkomycin biosynthetic gene cluster has been cloned from Streptomyces asochromogenes. Two cytochrome P450 monooxygenase genes (sanQ, sanH) and one ferredoxin gene (sanI) were found in the cluster. It was reported that SanQ is involved in the hydroxylation of l-His, a key step in 4-formyl-4-imidazolin-2-one base biosynthesis. Here, we have studied the function of sanH and sanI. Disruption of sanH abolished the production of nikkomycin X and Z, but it accumulated one dominant component nikkomycin Lx, which is the nikkomycin X analog lacking the hydroxy group at the pyridyl residue. The sanI disruption mutant accumulated predominantly nikkomycin Lx in addition to nikkomycin X and Z. The nikkomycin production profile of the sanH and sanI double disruption mutant was the same as that of the sanH disruption mutant. These results confirmed that SanH is essential for the hydroxylation of pyridyl residue in nikkomycin biosynthesis of S. ansochromogenes and first demonstrated that SanI is an effective electron donor for SanH, but not for SanQ in vivo.