Separation of peptide transport and hydrolysis in trimethionine uptake by Saccharomyces cerevisiae

Parker, David D.; Naider, Fred; Becker, Jeffrey M.

doi:10.1128/jb.143.2.1066-1069.1980

Cited by 8 publications

(6 citation statements)

References 14 publications

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“…In each case, the ratio of the activity in situ to that of the solubilized enzyme activity was less than 1.0. On the basis of reports (13,15,19) indicating that nitroanilide substrates are not readily transported across the cell membrane, the low activity ratios were commensurate with a more predominant cytoplasmic (rather than membrane) localization for the aminopeptidase activity. This was confirmed by the recovery of the major proportion of the total cellular aminopeptidase activity with the cytoplasmic fraction following ultracentrifugation of the protoplast lysates.…”

Section: Discussionmentioning

confidence: 90%

Comparison of aminopeptidase activities in four strains of mutans group oral streptococci

Cowman¹,

Baron²

1993

Infect Immun

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In this study, native cells of Streptococcus mutans VA-29R and Streptococcus rattus FA-1 displayed significantly higher aminopeptidase activity than did cells of Streptococcus cricetus AHT or Streptococcus sobrinus 6715 toward the nitroanilide derivatives of leucine, alanine, methionine, arginine, and lysine. These differences in cellular aminopeptidase activity led us to investigate the subcellular localization of the aminopeptidase in these mutans group streptococci. Following conversion of native cells to protoplasts by treatment with lysozyme, most of the aminopeptidase activity detected in the native-cell preparations remained associated with the intact protoplasts. After lysis of protoplasts and differential centrifugation, most of the total cellular aminopeptidase activity was recovered with the cytoplasmic fraction. Membrane-associated aminopeptidases represented only minor activities in these mutans group streptococci. Although the four strains showed no differences with respect to a predominant cytoplasmic localization for the aminopeptidase activities, the levels of activity in the cytoplasmic fractions from S. cricetus AHT and S. sobrinus 6715 were significantly lower than those measurable in the corresponding fractions from S. mutans VA-29R and S. rattus FA-1. These results support the conclusion that the differences in aminopeptidase activity expressed by these streptococci reflect quantitative differences rather than differences in enzyme subcellular localization.

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Section: Discussionmentioning

confidence: 90%

Comparison of aminopeptidase activities in four strains of mutans group oral streptococci

Cowman¹,

Baron²

1993

Infect Immun

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“…Early experiments on the utilization of short peptides by the budding yeast Saccharomyces cerevisiae were aimed at determining whether peptides were taken up by the cell directly or as amino acids after hydrolysis by extracellular peptidases. While some groups have reported weak extracellular aminopeptidase activity and suggested that it might be important for the utilization of amino acids from peptides (Frey and Rohm, 1978;Hirsch et al, 1988), other groups did not detect extracellular degradation of peptides (Parker et al, 1980;Nisbet and Payne, 1979). A few years later a broad-specificity transporter of di-and tripeptides from budding yeast was cloned (Perry et al, 1994); it was concluded that PTR2 is responsible for uptake and that the peptides are degraded intracellularly.…”

Section: Introductionmentioning

confidence: 99%

New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations

Melnykov

2015

Yeast

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The budding yeast Saccharomyces cerevisiae is able to take up large quantities of amino acids in the form of di-and tripeptides via a short peptide transporter, Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this upregulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids: the first is upregulation of PTR2 in a population of cells, caused by amino acid secretion that accompanies peptide uptake; the second is loss of Ptr2p activity, due to transporter internalization following peptide uptake. Our findings emphasize the importance of proper amino acid balance in the cell and extend understanding of peptide import regulation in yeast.

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“…In a previous study (Parker et al, 1980), we demonstrated that in the dark leucyl-p-nitroanilide (Leu-p-NA) is a competitive inhibitor of trimethionine transport in S. cerevisiae. Upon irradiation of cells in the presence of Leu-p-NA, trimethionine uptake was severely lowered (Figure 1).…”

Section: Resultsmentioning

confidence: 97%

“…However, at 1CT3 M, p-nitroaniline had no effect on peptide transport, and even photolysis of cells in the presence of high concentrations of p-nitroaniline (10~3 M) caused no effect on peptide uptake. Cells were irradiated in the presence of Leu-p-NA at 2.4 X 5 M, and the activity of cell-bound aminopeptidase was determined as described previously (Parker et al, 1980). Photolysis was carried out at pH 5.5 under the conditions described.…”

Section: Resultsmentioning

confidence: 99%

“…In a previous study (Parker et al, 1980), we showed that at pH 7.0 intact cells of S. cerevisiae 139 hydrolyzed leucyl-p-nitroanilide (Leu-p-NA)1 and other aminoacyl-p-nitroanilides by an activity similar to that of aminopeptidase II, a well-characterized external peptidase in yeast (Frey & Rohm, 1979). Furthermore, we showed that at pH 5.5 Leuf From the Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996 (J.M.B.…”

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confidence: 99%

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Photoinactivation of peptide transport in Saccharomyces cerevisiae

Becker¹,

Dunsmore²,

Steinfeld³

et al. 1982

Biochemistry

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Oligopeptides and dipeptides are transported into Saccharomyces cerevisiae by a carrier-mediated system. In the dark, leucyl-p-nitroanilide (Leu-p-NA) and leucyl-leucyl-4-azido-2-nitrophenylalanine [Leu-Leu-Phe-(4N3,2NO2)] are competitive inhibitors of peptide transport by S. cerevisiae cells. The photolysis of yeast cells in the presence of Leu-p-NA or Leu-Leu-Phe(4N3,2NO2) at 350 nm results in an irreversible inactivation of peptide transport. Protection against this inactivation is afforded by an excess of trimethionine, a transported peptide. Photolysis with Leu-p-NA or Leu-Leu-Phe(4N3,2NO2) does not affect amino acid or sugar transport, and cell viability is maintained throughout the irradiation procedure. A 5-min irradiation of S. cerevisiae with 2.4 microM Leu-p-NA or 15 microM Leu-Leu-Phe(4N3,2NO2) causes 50% inhibition of trimethionine uptake. p-Nitroaniline, a possible hydrolysis product generated from Leu-p-NA by cellular peptidase activity, has no effect on peptide transport. An exogenous energy source is not required for photoinactivation. The results suggest that a component(s) of the peptide transport system of S. cerevisiae is irreversibly modified by photolysis with Leu-p-NA or Leu-Leu-Phe-(4N3,2NO2) and provide the first example of the use of amino acid p-nitroanilides as photoaffinity labels.

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Separation of peptide transport and hydrolysis in trimethionine uptake by Saccharomyces cerevisiae

Cited by 8 publications

References 14 publications

Comparison of aminopeptidase activities in four strains of mutans group oral streptococci

Comparison of aminopeptidase activities in four strains of mutans group oral streptococci

New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations

Photoinactivation of peptide transport in Saccharomyces cerevisiae

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