Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Biochemistry and Cellular and Molecular Biology

Major Professor

Jeffrey M. Becker

Committee Members

Mary Ann Handel, Bruce McKee, Cynthia Peterson, Gary Stacey


The yeast Saccharomyces cerevisiae utilizes nutrient sensor activity at the plasma membrane to regulate growth. Amino acids are sensed by the membrane protein Ssy1p, and a signal is transduced to the novel intracellular regulatory protein Ptr3p. This leads to an upregulation of the expression of amino acid permease genes and PTR2, the gene encoding the di/tri-peptide transporter Ptr2p. Using a reporter gene assay, this study found that various amino acids induced PTR2 expression to different levels. Peptide and amino acid induction required Ptr3p, while Ssy1p was required for amino acid induction, but not peptide induction. Ptr3p-mediated, amino acid/dipeptide-induced expression of PTR2 did not involve transcriptional regulation of CUP9, a gene encoding a repressor of PTR2 expression. A functional chimeric protein formed by the fusion of GFP to the N-terminus of Ptr3p was found to be associated with the plasma membrane in a punctate pattern. A two-hybrid and co-immunoprecipitation analysis showed that Ptr3p interacted with the cytoplasmic, N-terminal 282 amino acid domain of Ssy1p further supporting the suggestion that Ssy1p/Ptr3p interaction was involved in generating a signal after amino acid sensing.

We also showed that STP1 and STP2, genes previously hypothesized to encode pre-tRNA splicing proteins and bind to the BAP2 promoter as transcription factors, were required for regulation of the di/tri-peptide transport system. The data provide the first evidence about the involvement of STP1 and STP2 in the di/tri-peptide transport system suggesting their function as transcription factors of PTR2. In contrast to the PTR2 expression pattern, in medium containing a rich nitrogen source STP1 and STP2 are expressed at higher levels in comparison to their expression in medium with a poor nitrogen source. STP2 likely acts downstream of PTR3 as shown by epistasis studies, and PTR3 does not regulate the expression of STP1 or STP2.

To provide insights into the function of Ptr3p, we performed a two-hybrid analysis to identify proteins that may interact with Ptr3p. We identified proteins involved in transport, transcription processes, and unknown functions suggesting that Ptr3p plays role in numerous pathways. We used two different computational algorithms to predict that Ptr3p contains two putative nuclear localization signals (NLS), and alanine substitution of one of these NLS decreased its function as a regulator of PTR2. PROSPECT, a 3-dimensional protein structure algorithm, also calculated that Ptr3p has a RING finger and a seven-bladded β-propeller motif. Alanine site-directed mutagenesis of some conserved residues in the ring finger motif reduced the function of Ptr3p. Mutations within the predicted β-propeller motif indicated that this motif may act as an autoinhibitory domain, because their substitution resulted in a more functional mutant than the wild type Ptr3p. Finally, we identified functionalm domains of Ptr3p. The expression of Ptr3p residues 1-250 or 1-500 partially complemented ptr3Δ phenotype, and residues 250-678 appeared to be as functional as full length Ptr3p. These results add further to an understanding of the role of Ptr3p in the amino acid induced signal transduction pathway.

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