Date of Award
Doctor of Philosophy
Biochemistry and Cellular and Molecular Biology
John Koontz, Pam Small, Cynthia Peterson, Mary Ann Handel
All cells require means to obtain nutrients, dispose of waste, and communicate with other cells. One mechanism fulfilling these requirements is active transport, and many permeases in proteomes provide this function. One family of permeases is the Oligopeptide Transport (OPT) family, which is characterized by transporting tetra/penta-peptides across the plasma membrane. In this research, an informatic approach was taken to identify 103 family members, all from plant or fungal species with sequence homology to OPTs of known function.
Eighteen conserved motifs were identified in these homologues. Using several computational algorithms and multiple sequence alignment, 12 transmembrane domains have been identified in the family, with the termini predicted to be outside of the cell.
Most, but not all, of the conserved motifs lie in the non-transmembrane loops of the proteins. One OPT family member in the yeast Saccharomyces cerevisiae, Opt1p, was further characterized and found to transport Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) and reduced glutathione (γ-Glu-Cys-Gly). The uptake of these substrates was inhibited by substrates of other OPTs (i.e. KLGL) and substrates that inhibit Leu-enkephalin uptake into mammalian brain (i.e.. naloxone, naltrexone, and morphine). A screen was conducted to identify an OPT functional homologue in rat brain, but none was found. The expression of the gene encoding Opt1p, OPT1, was characterized using a reporter construct with the promoter of OPT1 cloned up-stream of lacZ, the gene encoding β-galactosidase. When sulfur was absent from the medium, OPT1 was upregulated. Additionally, when naturally occurring amino acids were added to the sulfurfree medium, OPT1 was further up-regulated, up to 30-fold. Furthermore, this induction was dependent on the presence of PTR3 and SSY1, genes encoding Ptr3p and Ssy1p. These proteins form the SPS complex with Ssy5p to sense extracellular amino acids. The complex transduces the signal to up-regulate amino acid and the di/tri-peptide permease genes.
The OPT family is a relatively new family of transporters. This study of its structure, function, and regulation lays the groundwork and defines future areas of investigation.
Wiles, Amy Marie, "The Oligopeptide Transport Family: Informatic and Experimental Characterization. " PhD diss., University of Tennessee, 2005.