Doctoral Dissertations

Date of Award

12-1994

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Jeffery M. Becker

Committee Members

Mary Ann Handel, John Koontz, Stuart Riggsby

Abstract

The transport of small peptides across the plasma membrane, a phenomenon shown to occur in a range of procaryotes and eucaryotes, is mediated by specific proteins in an energy dependent fashion. Peptide transport systems have been well studied in prokaryotes, especially Escherichia coli and Salmonella typhimurium where several genes have been cloned and characterized. However information about the genes responsible for peptide transport in eucaryotes is just emerging. In Saccharomyces cerevisiae (baker's yeast) at least three genes are required for peptide transport: PTR1, PTR2 and PTR3. Molecular characterization of each of these genes in the model eucaryotic cell, S. cerevisiae, is central to our understanding of peptide transport in eucaryotes. The major goal of my project was to isolate and characterize one of these genes required for Peptide Transport in S. cerevisiae, namely PTR1. A review of current literature in peptide transport and the specific objectives leading to this research are covered in part I of this dissertation.

Part II of this dissertation describes the isolation of PTR1 from a S. cerevisiae genomic library by functional complementation of a peptide transport deficient ptr1 strain. This is followed by experiments which confirmed the restoration of peptide transport to the mutant strain to be a plasmid borne phenotype and not due to a reversion event in the mutant strain. Also described in this part are efforts to define the minimum length of the insert required for functional complementation and the physical mapping of PTR1 to chromosome VII of S. cerevisiae.

Part III of this dissertation documents findings that lead to the intriguing discovery that PTR1 is identical to UBRI, a gene described as the recognition component of the N- end rule portion of the ubiquitin system in S. cerevisiae. In this part, DNA sequence analysis of PTR1 and the phenotypic characterization of ptr1 and ubr1 mutant strains under relevant assay conditions are presented.

Part IV of this dissertation outlines efforts to elucidate the specific role of PTR1 in the peptide transport system of S. cerevisiae First, tests were performed to determine whether other genes of the ubiquitin system, such as the ubiquitin-conjugating enzymes (UBCs), were required for peptide transport. Results showed that those UBCs tested were not required for peptide transport in S. cerevisiae. Also presented in this part of the dissertation are results from a search of the predicted amino acid sequence of PTRI for specific motifs. The sequence analysis is followed by results from relevant transcriptional studies. The search for specific motifs in Ptrlp revealed several motifs which suggested a regulatory role for this protein. Northern analysis showed that Ptr1p is indeed a transcriptional regulator; Ptr1p is required for the transcription of PTR2, a structural component required for peptide transport in S. cerevisiae.

Part V of this dissertation presents two models which deals with Ptr1p as a transcriptional regulator. In the first model, this regulation is hypothesized to occur through a ubiquitin-mediated pathway. In the second model, the regulation is postulated to be independent of the ubiquitin system.

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