Doctoral Dissertations

Date of Award

3-1988

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

Jeffrey M. Becker

Committee Members

Leaf Huang, Frank Larimer, W. Stuart Riggsby

Abstract

In an effort to characterize the number, relative role and regulation of peptide transporters in Saccharomyces cerevisiae, the transport of a range of toxic and radiolabeled peptides was examined in this yeast under various physiological conditions. A new regulatory control on peptide transport was identified. The presence of micromolar concentrations of any one of numerous amino acids resulted in induction of pep tide transport activity. Increases in peptide transport were indicated by large increases in sensitivity to toxic peptides and by a 25-fold increase in the initial rate of tritiated leucyl-leucine uptake. Spontaneous, transport-deficient mutants were identified under induced conditions by their resistance to toxic di- or tripeptides and fell into three complementation groups. Such mutants accumulated radiolabeled peptides at minimal rates and lost only their amino acid-inducible sensitivity to toxic peptides. Each mutant retained the sensitivity to certain toxic dipeptides seen in the wild type strain in the absence of induction. The sensitivity to toxic peptides retained in peptide transport-deficient mutants may represent a second system for the utilization of some peptides or may be the result of periplasmic or membrane-associated peptidase activity. Since amino acid-induction of peptide transport activity was prevented by addition of cycloheximide, proteins synthesized in response to addition of an amino acid inducer were analysed on one- and two-dimensional polyacrylamide gels of pulse-labeled yeast extracts. A putative peptide permease could not be clearly identified, however, because none of the proteins synthesized in response to amino acid addition were also enriched in a crude yeast membrane fraction. Attempts at cloning the gene or genes which encode the peptide permease by complementation, or through use of a gene dosage selection, were unsuccessful.

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