JOURNAL OF BACTERIOLOGY, Dec. 1978, p. 1174-1177 0021-9193/78/0136-1174$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 36, No. 3 Printed in U.S.A.

NOTES Isolation of a Peptide Transport-Deficient Mutant of Yeast R. MARDER,' B. ROSE,' J. M. BECKER,2 AND F. NAIDER' * College of Staten Island, City University of New York, New York, New York 10301' and University of Tennessee, Knoxville, Tennessee 379162

Received for publication 23 August 1978

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-ethionylL-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 ["C]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.

The transport and utilization of peptides by bacteria, fungi, and mammalian gut have been extensively reported in the literature (2). Such studies have enumerated many criteria for peptide transport in each of these cells or tissues. For example in Escherichia coli, oligopeptides larger than pentapeptides do not enter the cell, and some peptides with D-residues are not transported as efficiently as their L-isomeric counterparts. The affinity for transport is also determined by the nature of the amine terminus of the peptide (4, 11, 12). In yeast, the amino acid sequence of a peptide appears to affect peptide uptake in several strains of Saccharomyces cerevisiae (8, 10). One very important aspect of characterizing peptide transport for any particular organism is to determine whether multiple transport systems exist for peptides in such an organism. In E. coli these studies have demonstrated two oligopeptide transport systems and a separate dipeptide transport system (3, 6, 9). In Neurospora crassa, only one general system has been found to mediate uptake of oligopeptides (14). The isolation and characterization of peptide transport mutants have been an invaluable aid in assaying the possible multiplicity of transport systems in these microorganisms. In S. cerevisiae G1333, a methionine auxotroph, transport studies demonstrated utilization of a wide variety of dipeptides and oligopeptides containing methionine (5, 10). Later studies with a lysine-leucine double auxotroph, S. cerevisiae Z1-2D, revealed similar characteristics of

peptide utilization (8). Many leucine dipeptides and tripeptides and several lysine peptides served as good sources of the required amino acids (Table 1). Competition studies between growth-essential, and non-growth-supporting peptides suggested that these dipeptides and tripeptides share a common mode of entry into this organism. The peptides enter the cell intact by a system independent of the transport of amino acids. However, from these studies alone it was not possible to conclude whether separate dipeptide and oligopeptide systems exist. It was therefore important to isolate and characterize a transport-deficient mutant of strain Z1-2D and compare its transport characteristics with those determined on the parent strain. S. cerevisiae Z1-2D is sensitive to the amino acid analogue L-ethionine. L-ethionyl-L-alanine and L-leucyl-L-leucyl-L-ethionine are equally toxic at the equivalent ethionine concentration of 0.23 mM. These peptides are probably toxic due to the release of L-ethionine by internal peptidases since a cell extract readily hydrolyzes L-ethionyl-L-alanine, and the toxicity of this peptide is prevented in the presence of a 7:1 molar excess of L-leucyl-L-leucine in the growth medium. Ames et al. (1) demonstrated that Lethionyl-L-alanine appears to enter Salmonella typhimurium through a dipeptide transport system. The following scheme was carried out to isolate a mutant resistant to L-ethionyl-L-alanine and possibly other peptides as well. Approximately 106 cells of a stationary culture of strain

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TABLE 1. Growth response' of strains Z1-2D and Z1-2D Etar Peptide

Amino acid(s)

Z1-2D

Z1-2D Etar

143 144 L-Leucine + L-lysine 2 2 L-Leucine 2 2 L-Lysine 2 110 L-Lysine L-Leucyl-L-leucine 2 103 L-Lysine L-Leucyl-L-leucyl-L-leucine 10 131 L-Lysine L-Leucyl-L-leucine benzyl ester 2 131 L-Leucine L-Lysyl-glycyl-glycine 2 2 L-Leucine + L-lysine + L-ethionine 154 4 L-Leucine + L-lysine L-Ethionyl-L-alanine 2 152 L-Leucine + L-lysine L-Leucyl-L-leucyl-L-ethionine a Growth as determined by increase in turbidity (Klett, blue filter) over a 40-h growth period. Klett values are normalized to an initial reading of 2. L-Leucine, L-ethionine, and related peptides were supplied in the liquid medium at an equivalent leucine concentration of 0.23 mM; L-lysine and L-lysyl-glycyl-glycine were supplied at 0.11 mM.

Z1-2D grown on 0.23 mM L-leucine, 0.11 mM L-lysine, 0.23 mM L-ethionyl-L-alanine, and 1.61 mM L-leucyl-L-leucine were plated out on a minimal medium agar plate containing L-leucine; Llysine, and L-ethionyl-L-alanine. A total of 13 colonies grew on this plate and were individually transferred to plates containing L-lysine and Lleucine; L-lysine and L-leucyl-L-leucine; and Llysine, L-leucine, and L-ethionine. Of the 13, 11 colonies had acquired a resistance to L-ethionine. Mutation to L-ethionine resistance has been found in previous studies (7). The two remaining colonies were still sensitive to L-ethionine, and did not grow on the plate containing L-leucyl-L-leucine. The colonies were tested further to demonstrate the requirements for L-leucine and L-lysine by streaking out on the appropriate plates. One colony, designated Z1-2D Etar, was subsequently transferred to a complete medium slant and stored at 4°C. Growth experiments in liquid culture with Z1-2D Etar verified the mutant's sensitivity to L-ethionine and its growth in the presence of L-ethionyl-L-alanine. Also, in contrast to the parent strain, Z1-2D Etar was resistant to the toxic tripeptide L-leucyl-Lleucyl-L-ethionine. As indicated in Table 1, growth experiments also confirmed that leucine and lysine dipeptides and tripeptides which serve as nutrients for the parent auxotroph were not utilized by the mutant. Nine other di- and tripeptides that were growth substrates for the parent strain were examined using crystal tests on leucine or lysine plates. In no case did any of these peptides support growth of Z1-2D Etar. Although it appears that two steps were used to select Z1-2D Etar, one should note that the first step of growing the parental cells with Lethionyl-L-alanine and an excess of L-leucyl-Lleucine is most probably coincidental with the success in obtaining the mutant. We believe that the actual selection occurred in the second step of this procedure when the cells were applied to

the plate containing L-ethionyl-L-alanine. Further evidence f9r a one-step selection comes from the characteristics of spontaneous revertants isolated from this mutant (see below). Cell extracts of S. cerevisiae Z1-2D and Z1-2D Etar were examined as to their relative abilities to cleave various peptides used in the growth experiments. The cell extracts were prepared as described previously (8), except that the buffer used in dialysis was 40 mM potassium phosphate (pH 7). The mutant cell extract released L-leucine and L-lysine from all non-growth-supporting peptides that were examined. For example, incubation of L-leucyl-L-leucine with mutant and parent cell extracts resulted in the release of nearly identical levels of L-leucine (Fig. 1). Quantitative peptidase assays with the fluorescent reagent o-phthalaldehyde (13) revealed that the mutant cell extract has a specific activity for hydrolyzing dileucine equal to the release of 0.10 ,imol of L-leucine per min per mg of protein, which is only about one-half the rate at which the parent cell extract cleaves this peptide (0.26 ,umol of L-leucine released per min per mg of protein). This reduced rate, however, was much higher than the rate at which L-lysyl-L-leucine, a good growth substrate for the parent, was hydrolyzed by the parent cell extract. These results allow us to suggest that the mutant's inability to utilize these peptides results from a failure of these peptides to be transported to the site of peptidase activity inside the cell. To confirm this hypothesis, the uptake of [14C]glycyl-L-leucine by strains Z1-2D and Zl2D Etar was examined. Uptake assays were performed on cells grown in yeast nitrogen base with proline as the nitrogen source in the presence of leucine and lysine. Control studies showed that both mutant and parent strains incorporated 1 yM [14C]methionine at nearly equivalent rates. Z1-2D also accumulated the label of 1 tiM [14C]glycyl-L-leucine (albeit at

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FIG. 1. High-voltage paper electrophoresis chromatogram of the hydrolysis products of L-leucyl-L-leucine (Leu-Leu) by Z1-2D and ZJ-2D Etar cell extracts (CE). Markers are represented by Leu (L-leucine, 100 pg) and Leu-Leu (156 pg). Each time point represents 50 ll of a 50:50 assay mixture of L-leucyl-L-leucine (6.5 mg/ml) and cell extract. Protein concentrations of the cell extracts were 0.39 mg/ml for S. cerevisiae Z1-2D and 0.30 mg/ml for S. cerevisiae Z1-2D Etar. Samples were incubated at 30°C, spotted on the chromatogram, and subjected to electrophoresis at pH 3.5 for 3 h at 3,000 V, and then stained with ninhydrin and fixed with a 1% solution of saturated Cu(NO)2 plus 0.2o HNO3 in a 2:1 mixture of acetone/ethanol.

lower rates than that for the uptake of methionine), but the mutant Z1-2D Etar did not take up any label from this peptide (no increase in counts during the assay period). To gain further information on the nature of the mutation in S. cerevisiae Z1-2D Etar, the frequency of reversion to growth on peptides was determined. Strain Z1-2D Etar was grown on minimal medium plus L-leucine and L-lysine for 24 h, and the cells were washed twice with and suspended in sterile distilled water. Approximately 3 x 106 to 5 x 106 cells were distributed on minimal medium plates containing L-lysine and either L-leucyl-L-leucine or L-leucyl-L-leucyl-L-leucine. The plates were incubated at 300C for 4 days, and the apparent revertant frequency was 4.3 x 10-6. All 40 revertant colonies that appeared were tested to determine their individual properties by replica plating onto appropriately supplemented minimal medium plates. Each revertant colony examined possessed the characteristics of the parental strain Z1-2D:

these colonies still required L-leucine and L-lysine for growth, were sensitive to L-ethionine and L-ethionyl-L-alanine, and grew on both Lleucyl-L-leucine and L-leucyl-L-leucyl-L-leucine. Therefore these revertants of strain Z1-2D Etar spontaneously regained the ability to transport dipeptides and tripeptides in one step. In conclusion, growth studies, peptidase assays, and radioactivity uptake studies show that S. cerevisiae Z1-2D Etar is a peptide transportdeficient mutant. Furthermore, since dipeptide and tripeptide uptake is simultaneously lost in this mutant and spontaneous revertants are able to grow on both di- and tripeptides, it is likely that these peptides share a general peptide transport system. This paper is submitted by R.M. and B.R. in partial fulfillment of the requirements for the Ph.D. degree at the City University of New York. We acknowledge support for this work by Public Health Service grants GM 22086 and GM 22087 from the National Institute of General Medical Sciences and a grant from the

VOL. 136, 1978 Faculty Research Award Program of City University of New York. J.M.B. and F.N. are Public Health Service Research Career Development Awardees GM 00094 and GM 00025 from the National Institute of General Medical Sciences.

LITERATURE CITED 1. Ames, B. N., G. F.-L. Ames, J. D. Young, D. Tsuchiya, and J. Lecocq. 1973. Illicit transport: the oligopeptide permease. Proc. Natl. Acad. Sci. U.S.A. 70:456-458. 2. Barak, Z., and C. Gilvarg. 1975. Peptide transport, p. 167-218. In H. Eisenberg, E. Katchalski-Katzir, and L. A. Manson (ed.), Biomembranes, vol. 7. Plenum Publishing Corp., New York. 3. Barak, Z., and C. Gilvarg. 1975. Specialized peptide transport system in Escherichia coli. J. Bacteriol. 122:1200-1207. 4. Becker, J. M., and F. Naider. 1974. Stereospecificity of tripeptide utilization in a methionine auxotroph of Escherichia coli K-12. J. Bacteriol. 120:191-196. 5. Becker, J. M., F. Naider, and E. Katchalski. 1973. Peptide utilization in yeast: studies on methionine and lysine auxotrophs of Saccharomyces cerevisiae. Biochim. Biophys. Acta 291:388-397. 6. DeFelice, M., J. Guardiola, A. Lamberti, and M. laccarino. 1975. Escherichia coli K-12 mutants altered in the transport systems for oligo- and dipeptides. J. Bac-

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teriol. 116:751-756. 7. Greer, H., and G. R. Fink. 1975. Isolation of regulatory mutants in Saccharomyces cerevisiae, p. 247-272. In D. Prescott (ed.), Methods in cell biology, vol. XI. Academic Press, New York. 8. Marder, R., J. M. Becker, and F. Naider. 1977. Peptide transport in yeast: utilization of leucine- and lysinecontaining peptides by Saccharomyces cerevisiae. J. Bacteriol. 131:906-916. 9. Naider, F., and J. M. Becker. 1975. Multiplicity of oligopeptide transport systems in Escherichia coli. J. Bacteriol. 122:1208-1215. 10. Naider, F., J. M. Becker, and E. Katzir-Katchalski. 1974. Utilization of methionine-containing peptides and their derivatives by a methionine requiring auxotroph of Saccharomyces cerevisiae. J. Biol. Chem. 249:9-20. 11. Payne, J. W. 1968. Oligopeptide transport in Escherichia coli: specificity with respect to side chain and distinction from dipeptide transport. J. Biol. Chem. 243:3395-3403. 12. Payne, J. W. 1971. The requirement for the protonated a-amino group for the transport of peptides in Escherichia coli. Biochem. J. 123:245-253. 13. Roth, M. 1971. Fluorescence reaction for amino acids. Anal. Chem. 43:880-882. 14. Wolfinbarger, L., Jr., and G. A. Marzluf. 1975. Stereospecificity and regulation of peptide transport in Neurospora crassa. Arch. Biochem. Biophys. 171:637-644.

Isolation of a peptide transport-deficient mutant of yeast.

JOURNAL OF BACTERIOLOGY, Dec. 1978, p. 1174-1177 0021-9193/78/0136-1174$02.00/0 Copyright © 1978 American Society for Microbiology Vol. 36, No. 3 Pri...
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