Doctoral Dissertations

Jack R. Perry

Date of Award

5-1995

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Jeffrey M. Becker

Committee Members

William S. Riggsby, Elizabeth E. Howell, Frank W. Larimer

Abstract

The transport of intact peptides across the plasma membrane is a well characterized physiological phenomenon known to occur in a variety of organisms. However, the genetic basis underlying this process is poorly understood in eukaryotic organisms. In order to better understand the genetic basis for transmembrane peptide transport, a group of S. cerevisiae strains deficient in the uptake of peptides was generated by chemical mutagenesis then characterized. Here it is shown that peptide transport in S. cerevisiae is mediated by the action of at least three genes: PTRI, PTR2, and PTR3. Strains carrying mutations in these genes define two phenotypic classes, designated A and B. S. cerevisiae strains of phenotypic class A, represented by Ptr1-; and Ptr2-; strains, are characterized by resistance to the growth inhibiting effects of toxic dipeptides, failure to accumulate radiolabeled dileucine at wildtype levels, and the inability to use dipeptides to satisfy auxotrophic amino acid requirements. S. cerevisiae strains of phenotypic class B, represented by Ptr3-; strains, are also characterized by resistance to the growth inhibiting effects of toxic dipeptides, but are able to accumulate low levels of radiolabeled dileucine, and are able to use a variety of dipeptides to satisfy amino acid growth requirements. In an effort to define peptide transport at the molecular level, methodologies for cloning genes involved in the various aspects of peptide transport in S. cerevisiae are presented. Also presented are results from the molecular cloning and characterization of the PTR2 gene of S. cerevisiae. The PTR2 gene was isolated from a YCp50-based genomic DNA library by directly selecting for functional complementation of the peptide transport deficient phenotype conferred by the ptr2-2 mutation. Deletion and frameshift mutagenesis were used to localize the complementing activity to a 3.1-kbp region on the transforming plasmid pJP2. DNA sequencing of the complementing region identified an open reading frame spanning 1803 bp. The deduced amino acid sequence predicts a hydrophobic peptide consisting of 601 amino acids with a molecular mass of 68.1-kDa. The predicted protein is composed in part of twelve hydrophobic segments similar to those found in proteins known to be plasma membrane associated and to function in transmembrane transport. Northern hybridization experiments demonstrate a single transcript of 1.8-kb in length commensurate with the size of the PTR2 open reading frame. The PTR2 encoded peptide was shown to share significant similarities with a nitrate inducible membrane protein, encoded by the CHLI gene, of Arabidopsis thaliana which functions in nitrate transport. A search of the DNA sequence data base showed the cloned PTR2 gene to be tightly linked to the UB12 gene with the coding sequences being separated by a 466-bpregion and oriented so that the genes are transcribed convergently. The PTR2 gene was localized to the right arm chromosome XI by CHEF gel chromosome blots and by hybridization to known chromosome XI λ phage clones of S. cerevisiae DNA. Chromosomal disruption of the PTR2 gene in a haploid strain was not lethal under standard growth conditions. The cloning of PTR2 represents the first example of the molecular genetic characterization of a eukaryotic oligopeptide transport gene and the second member of a new class of membrane associated proteins.

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